Beta-L-2&#39;-deoxy-nucleosides for the treatment of hepatitis B

ABSTRACT

This invention is directed to a method for treating a host infected with hepatitis B comprising administering an effective amount of an anti-HBV biologically active 2′-deoxy-β-L-erythro-pentofuranonucleoside or a pharmaceutically acceptable salt or prodrug thereof, wherein the 2′-deoxy-β-L-erythro-pentofuranonucleoside has the formula:  
                 
 
     wherein R is selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative; and BASE is a purine or pyrimidine base which may be optionally substituted. The 2′-deoxy-β-L-erythro-pentofuranonucleoside or a pharmaceutically acceptable salt or prodrug thereof may be administered either alone or in combination with another 2′-deoxy-β-L-erythro-pentofuranonucleoside or in combination with another anti-hepatitis B agent.

[0001] This application is a continuation in part of U.S. Ser. No.09/371,747 filed on Aug. 10, 1999, which claims priority to U.S.provisional applications U.S. Ser. No. 60/096,110, filed on Aug. 10,1998 and U.S. Ser. No. 60/131,352, filed on Apr. 28, 1999.

BACKGROUND OF THE INVENTION

[0002] This invention is in the area of methods for the treatment ofhepatitis B virus (also referred to as “HBV”) that includesadministering to a host in need thereof, either alone or in combination,an effective amount of one or more of the active compounds disclosedherein, or a pharmaceutically acceptable prodrug or salt of one of thesecompounds.

[0003] HBV is second only to tobacco as a cause of human cancer. Themechanism by which HBV induces cancer is unknown, although it ispostulated that it may directly trigger tumor development, or indirectlytrigger tumor development through chronic inflammation, cirrhosis, andcell regeneration associated with the infection.

[0004] Hepatitis B virus has reached epidemic levels worldwide. After atwo to six month incubation period in which the host is unaware of theinfection, HBV infection can lead to acute hepatitis and liver damage,that causes abdominal pain, jaundice, and elevated blood levels ofcertain enzymes. HBV can cause fulminant hepatitis, a rapidlyprogressive, often fatal form of the disease in which massive sectionsof the liver are destroyed.

[0005] Patients typically recover from acute hepatitis. In somepatients, however, high levels of viral antigen persist in the blood foran extended, or indefinite, period, causing a chronic infection. Chronicinfections can lead to chronic persistent hepatitis. Patients infectedwith chronic persistent HBV are most common in developing countries. Bymid-1991, there were approximately 225 million chronic carriers of HBVin Asia alone, and worldwide, almost 300 million carriers. Chronicpersistent hepatitis can cause fatigue, cirrhosis of the liver, andhepatocellular carcinoma, a primary liver cancer.

[0006] In western industrialized countries, high risk groups for HBVinfection include those in contact with HBV carriers or their bloodsamples. The epidemiology of HBV is very similar to that of acquiredimmune deficiency syndrome (AIDS), which accounts for why HBV infectionis common among patients with AIDS or AIDS related complex. However, HBVis more contagious than HIV.

[0007] However, more recently, vaccines have also been produced throughgenetic engineering and are currently used widely. Unfortunately,vaccines cannot help those already infected with HBV. Daily treatmentswith a-interferon, a genetically engineered protein, has also shownpromise, but this therapy is only successful in about one third oftreated patients. Further, interferon cannot be given orally.

[0008] A number of synthetic nucleosides have been identified whichexhibit activity against HBV. The (−)-enantiomer of BCH-189, known as3TC, claimed in U.S. Pat. No. 5,539,116 to Liotta, et al., has beenapproved by the U.S. Food and Drug Administration for the treatment ofhepatitis B. See also EPA 0 494 119 A1 filed by BioChem Pharma, Inc.

[0009] Cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(“FTC”) exhibits activity against HBV. See WO 92/15308; Furman, et al.,“The Anti-Hepatitis B Virus Activities, Cytotoxicities, and AnabolicProfiles of the (−) and (+) Enantiomers ofcis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]-Cytosine”Antimicrobial Agents and Chemotherapy, December 1992, page 2686-2692;and Cheng, et al., Journal of Biological Chemistry, Volume 267(20),13938-13942 (1992).

[0010] von Janta-Lipinski et al. disclose the use of the L-enantiomersof 3′-fluoro-modified β-2′-deoxyribonucleoside 5′-triphosphates for theinhibition of hepatitis B polymerases (J. Med. Chem., 1998,41,2040-2046). Specifically, the 5′-triphosphates of3′-deoxy-3′-fluoro-β-L-thymidine (β-L-FTTP),2′,3′-dideoxy-3′-fluoro-β-L-cytidine (β-L-FdCTP), and2′,3′-dideoxy-3′-fluoro-β-L-5-methylcytidine (β-L-FMethCTP) weredisclosed as effective inhibitors of HBV DNA polymerases.

[0011] WO 96/13512 to Genencor International, Inc. and Lipitek, Inc.discloses that certain L-ribofuranosyl nucleosides can be useful for thetreatment of cancer and viruses. Specifically disclosed is the use ofthis class of compounds for the treatment of cancer and HIV.

[0012] U.S. Pat. Nos. 5,565,438, 5,567,688 and 5,587,362 (Chu, et al.)disclose the use of 2′-fluoro-5-methyl-β-L-arabinofuranolyluridine(L-FMAU) for the treatment of hepatitis B and Epstein Barr virus.

[0013] Yale University and University of Georgia Research Foundation,Inc. disclose the use of L-FddC (β-L-5-fluoro-2′,3′-dideoxycytidine) forthe treatment of hepatitis B virus in WO 92/18517.

[0014] The synthetic nucleosides β-L-2′-deoxycytidine (β-L-2′-dC),β-L-2′-deoxythymidine (β-L-dT) and β-L-2′-deoxyadenosine (β-L-2′-dA),are known in the art. Antonin Holy first disclosed β-L-dC and β-L-dT in1972, “Nucleic Acid Components and Their Analogs. CLIII. Preparation of2′-deoxy-L-Ribonucleosides of the Pyrimidine Series,” Collect. Czech.Chem. Commun. (1972), 37(12), 4072-87. Morris S. Zedeck et al. firstdisclosed β-L-dA for the inhibition of the synthesis of induced enzymesin Pseudomonas testosteroni, Mol. Phys. (1967), 3(4), 386-95.

[0015] Certain 2′-deoxy-β-L-erythro-pentofuranonucleosides are known tohave antineoplastic and selected antiviral activities. Verri et al.disclose the use of 2′-deoxy-β-L-erythro-pentofuranonucleosides asantineoplastic agents and as anti-herpetic agents (Mol. Pharmacol.(1997), 51(1), 132-138 and Biochem. J. (1997), 328(1), 317-20).Saneyoshi et al. demonstrate the use of 2′-deoxy-L-ribonucleosides asreverse transcriptase (I) inhibitors for the control of retroviruses andfor the treatment of AIDS, Jpn. Kokai Tokkyo Koho JP06293645 (1994).

[0016] Giovanni et al. tested2′-deoxy-β-L-erythro-pentofuranonucleosides against partiallypseudorabies virus (PRV), Biochem. J. (1993), 294(2), 381-5.

[0017] Chemotherapeutic uses of2′-deoxy-β-L-erythro-pentofuranonucleosides were studied by Tyrsted etal. (Biochim. Biophys. Acta (1968), 155(2), 619-22) and Bloch, et al.(J. Med. Chem. (1967), 10(5), 908-12).

[0018] β-L-2′-deoxythymidine (β-L-dT) is known in the art to inhibitherpes simplex virus type 1 (HSV-1) thymidine kinase (TK). Iotti et al.,WO 92/08727, teaches that β-L-dT selectively inhibits thephosphorylation of D-thymidine by HSV-1 TK, but not by human TK.Spaldari et al. reported that L-thymidine is phosphorylated by herpessimplex virus type 1 thymidine kinase and inhibits viral growth, J. Med.Chem. (1992), 35(22), 4214-20.

[0019] In light of the fact that hepatitis B virus has reached epidemiclevels worldwide, and has severe and often tragic effects on theinfected patient, there remains a strong need to provide new effectivepharmaceutical agents to treat humans infected with the virus that havelow toxicity to the host.

[0020] Therefore, it is an object of the present invention to providenew methods and compositions for the treatment of human patients orother hosts infected with hepatitis B virus.

SUMMARY OF THE INVENTION

[0021] A method for the treatment of hepatitis B infection in humans andother host animals is disclosed that includes administering an effectiveamount of a biologically active2′-deoxy-β-L-erythro-pentofuranonucleoside (referred to alternativelyherein as a β-L-d-nucleoside or a β-L-2′-d-nucleoside) or apharmaceutically acceptable salt or prodrug thereof, administered eitheralone or in combination, optionally in a pharmaceutically acceptablecarrier. The term 2′-deoxy, as used in this specification, refers to anucleoside that has no substituent in the 2′-position.

[0022] The disclosed 2′-deoxy-β-L-erythro-pentofuranonucleosides, orpharmaceutically acceptable prodrugs or salts or pharmaceuticallyacceptable formulations containing these compounds are useful in theprevention and treatment of hepatitis B infections and other relatedconditions such as anti-HBV antibody positive and HBV-positiveconditions, chronic liver inflammation caused by HBV, cirrhosis, acutehepatitis, fulminant hepatitis, chronic persistent hepatitis, andfatigue. These compounds or formulations can also be usedprophylactically to prevent or retard the progression of clinicalillness in individuals who are anti-HBV antibody or HBV-antigen positiveor who have been exposed to HBV.

[0023] In one embodiment of the present invention, the2′-deoxy-β-L-erythro-pentofuranonucleoside derivative is a compound ofthe formula:

[0024] wherein R is selected from the group consisting of H, straightchained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl,CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or aphosphate derivative; and BASE is a purine or pyrimidine base which mayoptionally be substituted.

[0025] In another embodiment, the2′-deoxy-β-L-erythro-pentofuranonucleoside derivative isβ-L-2′-deoxyadenosine or a pharmaceutically acceptable salt or prodrugthereof, of the formula:

[0026] wherein R is H, mono, di or tri phosphate, amino acid residue,acyl, or alkyl, or a stabilized phosphate derivative (to form astabilized nucleotide prodrug).

[0027] In another embodiment, the2′-deoxy-β-L-erythro-pentofuranonucleoside derivative isβ-L-2′-deoxycytidine or pharmaceutically acceptable salt or prodrugthereof of the formula:

[0028] wherein R is H, mono, di or tri phosphate, amino acid residue,acyl, or alkyl, or a stabilized phosphate derivative (to form astabilzied nucleotide prodrug).

[0029] In another embodiment, the2′-deoxy-β-L-erythro-pentofuranonucleoside derivative isβ-L-2′-deoxyuridine or pharmaceutically acceptable salt or prodrugthereof of the formula:

[0030] wherein R is H, mono, di or tri phosphate, amino acid residue,acyl, or alkyl, or a stabilized phosphate derivative (to form astabilzied nucleotide prodrug).

[0031] In another embodiment, the2′-deoxy-β-L-erythro-pentofuranonucleoside derivative isβ-L-2′-deoxyguanosine or pharmaceutically acceptable salt or prodrugthereof of the formula:

[0032] wherein R is H, mono, di or tri phosphate, amino acid residue,acyl, or alkyl, or a stabilized phosphate derivative (to form astabilized nucleotide prodrug).

[0033] In another embodiment, the2′-deoxy-β-L-erythro-pentofuranonucleoside derivative isβ-L-2′-deoxyinosine or pharmaceutically acceptable salt or prodrugthereof of the formula:

[0034] wherein R is H, mono, di or tri phosphate, amino acid residue,acyl, or alkyl, or a stabilized phosphate derivative (to form astabilized nucleotide prodrug).

[0035] In another embodiment, the2′-deoxy-β-L-erythro-pentofuranonucleoside derivative is β-L-thymidineor a pharmaceutically acceptable salt or prodrug thereof of the formula:

[0036] wherein R is H, mono, di or tri phosphate, amino acid residue,acyl, or alkyl, or a stabilized phosphate derivative (to form astabilized nucleotide prodrug).

[0037] In another embodiment, the2′-deoxy-β-L-erythro-pentofuranonucleoside is administered inalternation or combination with one or more other2′-deoxy-β-L-erythro-pentofuranonucleosides or one or more othercompounds which exhibit activity against hepatitis B virus. In general,during alternation therapy, an effective dosage of each agent isadministered serially, whereas in combination therapy, an effectivedosage of two or more agents are administered together. The dosages willdepend on absorption, inactivation, and excretion rates of the drug aswell as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens and schedules should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions.

[0038] In another embodiment, the invention includes a method for thetreatment of humans infected with HBV that includes administering an HBVtreatment amount of a prodrug of the disclosed2′-deoxy-β-L-erythro-pentofuranonucleoside derivatives. A prodrug, asused herein, refers to a compound that is converted into the nucleosideon administration in vivo. Nonlimiting examples include pharmaceuticallyacceptable salt (alternatively referred to as “physiologicallyacceptable salts”), the 5′ and N⁴ (cytidine) or N⁶ (adenosine) acylated(including with an amino acid residue such as L-valinyl) or alkylatedderivatives of the active compound, or the 5′-phospholipid or 5′-etherlipids of the active compound.

BRIEF DESCRIPTION OF THE FIGURE

[0039]FIG. 1 illustrates a general process for obtainingβ-L-erythro-pentafuranonucleosides (β-L-dN) using L-ribose or L-xyloseas a starting material.

[0040]FIG. 2 is a graph which illustrates the metabolism of L-dA, L-dC,and L-dT in human Hep G2 cells in terms of accumulation and decay. Thecells were incubated with 10 μM of compound.

[0041]FIG. 3 is a graph which illustrates the antiviral effect ofβ-L-dA, β-L-dT and β-L-dC in the woodchuck chronic hepatitis model.

DETAILED DESCRIPTION OF THE INVENTION

[0042] As used herein, the term “substantially in the form of a singleisomer” or “in isolated form” refers to a2′-deoxy-β-L-erythro-pentofuranonucleoside that is at leastapproximately 95% in the designated stereoconfiguration. In a preferredembodiment, the active compound is administered in at least this levelof purity to the host in need of therapy.

[0043] As used herein, the term hepatitis B and related conditionsrefers to hepatitis B and related conditions such as anti-HBV antibodypositive and HBV-positive conditions, chronic liver inflammation causedby HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chronicpersistent hepatitis, and fatigue. The method of the present inventionincludes the use of 2′-deoxy-β-L-erythro-pentofuranonucleosidederivatives prophylactically to prevent or retard the progression ofclinical illness in individuals who are anti-HBV antibody or HBV-antigenpositive or who have been exposed to HBV.

[0044] As used herein, the term alkyl, unless otherwise specified,refers to a saturated straight, branched, or cyclic, primary, secondary,or tertiary hydrocarbon, typically of C₁ to C₁₉, preferably C₁ to C₆ andspecifically includes but is not limited to methyl, ethyl, propyl,butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, t-butyl,isopentyl, amyl, t-pentyl, cyclopentyl, and cyclohexyl.

[0045] As used herein, the term acyl refers to moiety of the formula—C(O)R′, wherein R′ is alkyl; aryl, alkaryl, aralkyl, amino acidresidue, heteroaromatic, alkoxyalkyl including methoxymethyl; arylalkylincluding benzyl; aryloxyalkyl such as phenoxymethyl; aryl includingphenyl optionally substituted with halogen, C₁ to C₄alkyl or C₁ toC₄alkoxy, or the residue of an amino acid. The term acyl specificallyincludes but is not limited to acetyl, propionyl, butyryl, pentanoyl,3-methylbutyryl, hydrogen succinate, 3-chlorobenzoate, benzoyl, acetyl,pivaloyl, mesylate, propionyl, valeryl, caproic, caprylic, capric,lauric, myristic, palmitic, stearic, and oleic.

[0046] As used herein, the term purine or pyrimidine base, includes, butis not limited to, 6-alkylpurine and N⁶-alkylpurines, N⁶-acylpurines,N⁶-benzylpurine, 6-halopurine, N⁶-vinylpurine, N⁶-acetylenic purine,N⁶-acyl purine, N⁶-hydroxyalkyl purine, N⁶-thioalkyl purine,N²-alkylpurines, N⁴-alkylpyrimidines, N⁴-acylpyrimidines,4-benzylpyrimidine, N⁴-halopyrimidines, N⁴-acetylenic pyrimidines,4-acyl and N⁴-acyl pyrimidines, 4-hydroxyalkyl pyrimidines, 4-thioalkylpyrimidines, thymine, cytosine, 6-azapyrimidine, including6-azacytosine, 2- and/or 4-mercaptopyrimidine, uracil,C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines,C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine,C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine,C⁵-nitropyrimidine, C⁵-aminopyrimidine, N²-alkylpurines,N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl. Functional oxygen and nitrogen groups on the basecan be protected as necessary or desired. Suitable protecting groups arewell known to those skilled in the art, and include trimethylsilyl,dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl,trityl, alkyl groups, acyl groups such as acetyl and propionyl,methanesulfonyl, and p-toluenesulfonyl.

[0047] As used herein, the term amino acid residue includes but is notlimited to the L or D enantiomers (or any mixture thereof, including aracemic mixture)of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl,phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl,glutaoyl, lysinyl, argininyl, and histidinyl. Preferred amino acids arein the L-stereoconfiguration, and a preferred amino acid moiety isL-valinyl.

[0048] The term biologically active nucleoside, as used herein, refersto a nucleoside which exhibits an EC₅₀ of 15 micromolar or less whentested in 2.2.15 cells transfected with the hepatitis virion.

[0049] Preferred bases include cytosine, 5-fluorocytosine,5-bromocytosine, 5-iodocytosine, uracil, 5-fluorouracil, 5-bromouracil,5-iodouracil, 5-methyluracil, thymine, adenine, guanine, inosine,xanthine, 2,6-diaminopurine, 6-aminopurine, 6-chloropurine and2,6-dichloropurine, 6-bromopurine, 2,6-dibromopurine, 6-iodopurine,2,6-di-iodopurine, 5-bromovinylcytosine, 5-bromovinyluracil,5-bromoethenylcytosine, 5-bromoethenyluracil, 5-trifluoromethylcytosine,5-trifluoromethyluracil.

[0050] The 2′-deoxy-β-L-erythro-pentofuranonucleoside can be provided asa 5′ phospholipid or a 5′-ether lipid, as disclosed in the followingreferences, which are incorporated by reference herein: Kucera, L. S.,N. Lyer, E. Leake, A. Raben, Modest E. J., D. L. W., and C. Piantadosi.1990. Novel membrane-interactive ether lipid analogs that inhibitinfectious HIV-1 production and induce defective virus formation. AIDSRes Hum Retroviruses. 6:491-501; Piantadosi, C., J. Marasco C. J., S. L.morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq, L. S.Kucera, N. lyer, C. A. Wallen, S. Piantadosi, and E. J. Modest.1991-Synthesis and evaluation of novel ether lipid nucleoside conjugatesfor anti-HIV activity. J Med Chem. 34:1408-1414; Hostetler, K. Y., D. D.Richman, D. A. Carson, L. M. Stuhmiller, G. M. T. van Wijk, and H. vanden Bosch. 1992. Greatly enhanced inhibition of human immunodeficiencyvirus type 1 replication in CEM and HT4-6C cells by 31-deoxythymidinediphosphate dimyristoylglycerol, a lipid prodrug of 31-deoxythymidine.Antimicrob Agents Chemother. 36:2025-2029; Hostetler, K. Y., L. M.Stuhmiller, H. B. Lenting, H. van den Bosch, and D. D. Richman. 1990.Synthesis and antiretroviral activity of phospholipid analogs ofazidothymidine and other antiviral nucleosides. J. Biol Chem.265:6112-7.

[0051] The 2′-deoxy-β-L-erythro-pentofuranonucleoside can be convertedinto a pharmaceutically acceptable ester by reaction with an appropriateesterifying agent, for example, an acid halide or anhydride. Thenucleoside or its pharmaceutically acceptable prodrug can be convertedinto a pharmaceutically acceptable salt thereof in a conventionalmanner, for example, by treatment with an appropriate base or acid. Theester or salt can be converted into the parent nucleoside, for example,by hydrolysis.

[0052] As used herein, the term pharmaceutically acceptable salts orcomplexes refers to salts or complexes of the2′-deoxy-β-L-erythro-pentofuranonucleosides that retain the desiredbiological activity of the parent compound and exhibit minimal, if any,undesired toxicological effects. Nonlimiting examples of such salts are(a) acid addition salts formed with inorganic acids (for example,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid, and the like), and salts formed with organic acids such asacetic acid, oxalic acid, tartaric acid, succinic acid, malic acid,ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonicacids, and polygalacturonic acid; (b) base addition salts formed withcations such as sodium, potassium, zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium,and the like, or with an organic cation formed fromN,N-dibenzylethylene-diamine, ammonium, or ethylenediamine; or (c)combinations of (a) and (b); e.g., a zinc tannate salt or the like.

[0053] The term prodrug, as used herein, refers to a compound that isconverted into the nucleoside on administration in vivo. Nonlimitingexamples are pharmaceutically acceptable salts (alternatively referredto as “physiologically acceptable salts”), the 5′ and N⁴ or N⁶acylatedor alkylated derivatives of the active compound, and the 5′-phospholipidand 5′-ether lipid derivatives of the active compound.

[0054] Modifications of the active compounds, specifically at the N⁴, N⁶and 5′-O positions, can affect the bioavailability and rate ofmetabolism of the active species, thus providing control over thedelivery of the active species. A preferred modification is a 5′-aminoacid ester, including the L-valinyl ester.

[0055] A preferred embodiment of the present invention is a method forthe treatment of HBV infections in humans or other host animals, thatincludes administering an effective amount of one or more of a2′-deoxy-β-L-erythro-pentofuranonucleoside derivative selected from thegroup consisting of β-L-2′-deoxyadenosine, β-L-2′-deoxycytidine,β-L-2′-deoxyuridine, β-L-2′-guanosine, β-L-2′-deoxyinosine, andD-L-2′-deoxythymidine, or a physiologically acceptable prodrug thereof,including a phosphate, 5′ and or N⁶alkylated or acylated derivative, ora physiologically acceptable salt thereof, optionally in apharmaceutically acceptable carrier. The compounds of this inventioneither possess anti-HBV activity, or are metabolized to a compound orcompounds that exhibit anti-HBV activity. In a preferred embodiment, the2′-deoxy-β-L-erythro-pentofuranonucleoside is administered substantiallyin the form of a single isomer, i.e., at least approximately 95% in thedesignated stereoconfiguration.

[0056] Nucleotide Prodrugs

[0057] Any of the nucleosides described herein can be administered as astabilized nucleotide prodrug to increase the activity, bioavailability,stability or otherwise alter the properties of the nucleoside. A numberof nucleotide prodrug ligands are known. In general, alkylation,acylation or other lipophilic modification of the mono, di ortriphosphate of the nucleoside will increase the stability of thenucleotide. Examples of substituent groups that can replace one or morehydrogens on the phosphate moiety are alkyl, aryl, steroids,carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Manyare described in R. Jones and N. Bischofberger, Antiviral Research, 27(1995) 1-17. Any of these can be used in combination with the disclosednucleosides to achieve a desired effect.

[0058] In one embodiment, the 2′-deoxy-β-L-erythro-pentofuranonucleosideis provided as 5′-hydroxyl lipophilic prodrug. Nonlimiting examples ofU.S. patents that disclose suitable lipophilic substituents that can becovalently incorporated into the nucleoside, preferably at the 5′-OHposition of the nucleoside or lipophilic preparations, include U.S. Pat.Nos. 5,149,794 (Sep. 22, 1992, Yatvin et al.); 5,194,654 (Mar. 16, 1993,Hostetler et al., 5,223,263 (Jun. 29, 1993, Hostetler et al.); 5,256,641(Oct. 26, 1993, Yatvin et al.); 5,411,947 (May 2, 1995, Hostetler etal.); 5,463,092 (Oct. 31, 1995, Hostetler et al.); 5,543,389 (Aug. 6,1996, Yatvin et al.); 5,543,390 (Aug. 6, 1996, Yatvin et al.); 5,543,391(Aug. 6, 1996, Yatvin et al.); and 5,554,728 (Sep. 10, 1996; Basava etal.), all of which are incorporated herein by reference.

[0059] Foreign patent applications that disclose lipophilic substituentsthat can be attached to the 2′-deoxy-β-L-erythro-pentofuranonucleosidederivative of the present invention, or lipophilic preparations, includeWO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.

[0060] Additional nonlimiting examples of2′-deoxy-β-L-erythro-pentofuranonucleosides are those that containsubstituents as described in the following publications. Thesederivatized 2′-deoxy-β-L-erythro-pentofuranonucleosides can be used forthe indications described in the text or otherwise as antiviral agents,including as anti-HBV agents. Ho, D. H. W. (1973) Distribution of kinaseand deaminase of 1 β-D-arabinofuranosylcytosine in tissues of man andmouse. Cancer Res. 33, 2816-2820; Holy, A. (1993) Isopolarphosphorous-modified nucleotide analogues. In: De Clercq (Ed.), Advancesin Antiviral Drug Design, Vol. I, JAI Press, pp. 179-231; Hong, C. I.,Nechaev, A., and West, C. R. (1979a) Synthesis and antitumor activity of1□-D-arabinofuranosylcytosine conjugates of cortisol and cortisone.Biochem. Biophys. Rs. Commun. 88, 1223-1229; Hong, C. I., Nechaev, A.,Kirisits, A. J. Buchheit, D. J. and West, C. R. (1980) Nucleosideconjugates as potential antitumor agents. 3. Synthesis and antitumoractivity of 1-(β-D-arabinofuranosyl)cytosine conjugates ofcorticosteriods and selected lipophilic alcohols. J. Med. Chem. 28,171-177; Hostetler, K. Y., Stuhmiller, L. M., Lenting, H. B. M. van denBosch, H. and Richman, D. D. (1990) Synthesis and antiretroviralactivity of phospholipid analogs of azidothymidine and other antiviralnucleosides. J. Biol. Chem. 265, 6112-6117; Hostetler, K. Y., Carson, D.A. and Richman, D. D. (1991); Phosphatidylazidothymidine: mechanism ofantiretroviral action in CEM cells. J. Biol. Chem. 266, 11714-11717;Hostetler, K. Y., Korba, B. Sridhar, C., Gardener, M. (1994a) Antiviralactivity of phosphatidyl-dideoxycytidine in hepatitis B-infected cellsand enhanced hepatic uptake in mice. Antiviral Res. 24, 59-67;Hostetler, K. Y., Richman, D. D., Sridhar, C. N. Felgner, P. L, Felgner,J., Ricci, J., Gardener, M. F. Selleseth, D. W. and Ellis, M. N. (1994b)Phosphatidylazidothymidine and phosphatidyl-ddC: Assessment of uptake inmouse lymphoid tissues and antiviral activities in humanimmunodeficiency virus-infected cells and in rauscher leukemiavirus-infected mice. Antimicrobial Agents Chemother. 38, 2792-2797;Hunston, R. N., Jones, A. A. McGuigan, C., Walker, R. T., Balzarini, J.,and De Clercq, E. (1984) Synthesis and biological properties of somecyclic phosphotriesters derived from 2′-deoxy-5-fluorouridine. J. Med.Chem. 27, 440-444; Ji, Y. H., Moog, C., Schmitt, G., Bischoff, P. andLuu, B. (1990); Monophosphoric acid diesters of 7β-hydroxycholesteroland of pyrimidine nucleosides as potential antitumor agents: synthesisand preliminary evaluation of antitumor activity. J. Med. Chem. 33,2264-2270; Jones, A. S., McGuigan, C., Walker, R. T., Balzarini, J. andDeClercq, E. (1984) Synthesis, properties, and biological activity ofsome nucleoside cyclic phosphoramidates. J. Chem. Soc. Perkin Trans. I,1471-1474; Juodka, B. A. and Smart, J. 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(1989) Antitumor activity and pharmacology of1-β-D-arabinofuranosylcytosine-5′-stearylphosphate; an orally activederivative of 1-β-D-arabinofuranosylcytosine. Jpn. J. Cancer Res. 80,679-685; Korty, M. and Engels, J. (1979) The effects of adenosine- andguanosine 3′,5′-phosphoric and acid benzyl esters on guinea-pigventricular myocardium. Naunyn-Schmiedeberg's Arch. Pharmacol. 310,103-111; Kumar, A., Goe, P. L., Jones, A. S. Walker, R. T. Balzarini, J.and De Clercq, E. (1990) Synthesis and biological evaluation of somecyclic phosphoramidate nucleoside derivatives. J. Med. Chem. 33,2368-2375; LeBec, C., and Huynh-Dinh, T. (1991) Synthesis of lipophilicphosphate triester derivatives of 5-fluorouridine and arabinocytidine asanticancer prodrugs. Tetrahedron Lett. 32,6553-6556; Lichtenstein, J.,Barner, H. D. and Cohen, S. S. (1960) The metabolism of exogenouslysupplied nucleotides by Escherichia coli., J. Biol. Chem. 235, 457-465;Lucthy, J., Von Daeniken, A., Friederich, J. Manthey, B., Zweifel, J.,Schlatter, C. and Benn, M. H. (1981) Synthesis and toxicologicalproperties of three naturally occurring cyanoepithioalkanes. Mitt. Geg.Lebensmittelunters. Hyg. 72, 131-133 (Chem. Abstr. 95, 127093);McGuigan, C. Tollerfield, S. M. and Riley, P. A. (1989) Synthesis andbiological evaluation of some phosphate triester derivatives of theanti-viral drug Ara. Nucleic Acids Res. 17, 6065-6075; McGuigan, C.,Devine, K. G., O'Connor, T. J., Galpin, S. A., Jeffries, D. J. andKinchington, D. (1990a) Synthesis and evaluation of some novelphosphoramidate derivatives of 3′-azido-3′-deoxythymidine (AZT) asanti-HIV compounds. Antiviral Chem. Chemother-1, 107-113; McGuigan, C.,O'Connor, T. J., Nicholls, S. R. Nickson, C. and Kinchington, D. (1990b)Synthesis and anti-HIV activity of some novel substituted dialkylphosphate derivatives of AZT and ddCyd. Antiviral Chem. Chemother. 1,355-360; McGuigan, C., Nicholls, S. R., O'Connor, T. J., andKinchington, D. (1990c) Synthesis of some novel dialkyl phosphatederivative of 3′-modified nucleosides as potential anti-AIDS drugs.Antiviral Chem. Chemother. 1, 25-33; McGuigan, C., Devine, K. G.,O'Connor, T. J., and Kinchington, D.(1991) Synthesis and anti-HIVactivity of some haloalkyl phosphoramidate derivatives of3′-azido-3′-deoxythymidine (AZT); potent activity of the trichloroethylmethoxyalaninyl compound. Antiviral Res. 15, 255-263; McGuigan, C.,Pathirana, R. N., Mahmood, N., Devine, K. G. and Hay, A. J. (1992) Arylphosphate derivatives of AZT retain activity against HIV-1 in cell lineswhich are resistant to the action of AZT. Antiviral Res. 17, 311-321;McGuigan, C., Pathirana, R. N., Choi, S. M., Kinchington, D. andO'Connor, T. J. (1993a) Phosphoramidate derivatives of AZT as inhibitorsof HIV; studies on the carboxyl terminus. Antiviral Chem. Chemother. 4,97-101; McGuigan, C., Pathirana, R. N., Balzarini, J. and De Clercq, E.(1993b) Intracellular delivery of bioactive AZT nucleotides by arylphosphate derivatives of AZT. J. Med. Chem. 36, 1048-1052.

[0061] The question of chair-twist equilibria for the phosphate rings ofnucleoside cyclic 3′,5′-monophosphates. ¹HNMR and x-ray crystallographicstudy of the diasteromers of thymidine phenyl cyclic3′,5′-monophosphate. J. Am. Chem. Soc. 109, 4058-4064; Nerbonne, J. M.,Richard, S., Nargeot, J. and Lester, H. A. (1984) New photoactivatablecyclic nucleotides produce intracellular jumps in cyclic AMP and cyclicGMP concentrations. Nature 301, 74-76; Neumann, J. M., Hervé, M.,Debouzy, J. C., Guerra, F. I., Gouyette, C., Dupraz, B. and Huynh-Dinh,T. (1989) Synthesis and transmembrane transport studies by NMR of aglucosyl phospholipid of thymidine. J. Am. Chem. Soc. 111, 4270-4277;Ohno, R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H., Nakamura, T.,Kosaka, M., Takatuski, K., Yamaya, T., Toyama, K., Yoshida, T., Masaoka,T., Hashimoto, S., Ohshima, T., Kimura, I., Yamada, K. and Kimura, J.(1991) Treatment of myelodysplastic syndromes with orally administered1-β-D-rabinofuranosylcytosine-5′-stearylphosphate. Oncology 48, 451-455.

[0062] Palomino, E., Kessle, D. and Horwitz, J. P. (1989) Adihydropyridine carrier system for sustained delivery of2′,3′-dideoxynucleosides to the brain. J. Med. Chem. 32, 622-625;Perkins, R. M., Barney, S., Wittrock, R., Clark, P. H., Levin, R.Lambert, D. M., Petteway, S. R., Serafinowska, H. T., Bailey, S. M.,Jackson, S., Harnden, M. R. Ashton, R., Sutton, D., Harvey, J. J. andBrown, A. G. (1993) Activity of BRL47923 and its oral prodrug, SB203657Aagainst a rauscher murine leukemia virus infection in mice. AntiviralRes. 20 (Suppl. I). 84; Piantadosi, C., Marasco, C. J., Jr.,Morris-Natschke, S. L., Meyer, K. L., Gumus, F., Surles, J. R., Ishaq,K. S., Kucera, L. S. Iyer, N., Wallen, C. A., Piantadosi, S. and Modest,E. J. (1991) Synthesis and evaluation of novel ether lipid nucleosideconjugates for anti-HIV-1 activity. J. Med. Chem. 34, 1408-1414; Pompon,A., Lefebvre, I., Imbach, J. L., Kahn, S. and Farquhar, D. (1994)Decomposition pathways of the mono- and bis(pivaloyloxymethyl) esters ofazidothymidine-5′-monophosphate in cell extract and in tissue culturemedium; an application of the ‘on-line ISRP-cleaning’ HPLC technique.Antiviral Chem. Chemother. 5, 91-98; Postemark, T. (1974) Cyclic AMP andcyclic GMP. Annu. Rev. Pharmacol. 14, 23-33; Prisbe, E. J., Martin, J.C. M., McGee, D. P. C., Barker, M. F., Smee, D. F. Duke, A. E.,Matthews, T. R. and Verheyden, J. P. J. (1986) Synthesis and antiherpesvirus activity of phosphate and phosphonate derivatives of9-[(1,3-dihydroxy-2-propoxy)methyl] guanine. J. Med. Chem. 29, 671-675;Puech, F., Gosselin, G., Lefebvre, I., Pompon, A., Aubertin, A. M. Dim,A. and Imbach, J. L. (1993) Intracellular delivery of nucleosidemonophosphate through a reductase-mediated activation process. AntiviralRes. 22, 155-174; Pugaeva, V. P., Klochkeva, S. I., Mashbits, F. D. andEizengart, R. S. (1969). Robins, R. K. (1984) The potential ofnucleotide analogs as inhibitors of retroviruses and tumors. Pharm. Res.11-18; Rosowsky, A., Kim. S. H., Ross and J. Wick, M. M. (1982)Lipophilic 5′-(alkylphosphate) esters of 1-β-D-arabinofuranosylcytosineand its N⁴-acyl and 2.2′-anhydro-3′-O-acyl derivatives as potentialprodrugs. J. Med. Chem. 25, 171-178; Ross, W. (1961) Increasedsensitivity of the walker turnout towards aromatic nitrogen mustardscarrying basic side chains following glucose pretreatment. Biochem.Pharm. 8, 235-240; Ryu, E. K., Ross, R. J. Matsushita, T., MacCoss, M.,Hong, C. I. and West, C. R. (1982). Phospholipid-nucleoside conjugates.3. Synthesis and preliminary biological evaluation of1-β-D-arabinofuranosylcytosine 5′diphosphate[−], 2-diacylglycerols. J.Med. Chem. 25, 1322-1329; Saffhill, R. and Hume, W. J. (1986) Thedegradation of 5-iododeoxyuridine and 5-bromodeoxyuridine by serum fromdifferent sources and its consequences for the use of these compoundsfor incorporation into DNA. Chem. Diol. Interact. 57, 347-355;Saneyoshi, M., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. andYoshino, H. (1980) Synthetic nucleosides and nucleotides. XVI. Synthesisand biological evaluations of a series of 1-β-D-arabinofuranosylcytosine5′-alkyl or arylphosphates. Chem. Pharm. Bull. 28, 2915-2923; Sastry, J.K., Nehete, P. N., Khan, S., Nowak, B. J., Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1992) Membrane-permeable dideoxyuridine5′-monophosphate analogue inhibits human immunodeficiency virusinfection. Mol. Pharmacol. 41, 441-445; Shaw, J. P., Jones, R. J.Arimilli, M. N., Louie, M. S., Lee, W. A. and Cundy, K. C. (1994) Oralbioavailability of PMEA from PMEA prodrugs in male Sprague-Dawley rats.9th Annual AAPS Meeting. San Diego, Calif. (Abstract). Shuto, S., Ueda,S., Imamura, S., Fukukawa, K. Matsuda, A. and Ueda, T. (1987) A facileone-step synthesis of 5′-phosphatidylnucleosides by an enzymatictwo-phase reaction. Tetrahedron Lett. 28, 199-202; Shuto, S., Itoh, H.,Ueda, S., Imamura, S., Kukukawa, K., Tsujino, M., Matsuda, A. and Ueda,T. (1988) A facile enzymatic synthesis of5′-(3-sn-phosphatidyl)nucleosides and their antileukemic activities.Chem. Pharm. Bull. 36, 209-217. One preferred phosphate prodrug group isthe S-acyl-2-thioethyl group, also referred to as “SATE.”

[0063] Combination or Alternation Therapy

[0064] It has been recognized that drug-resistant variants of HBV canemerge after prolonged treatment with an antiviral agent. Drugresistance most typically occurs by mutation of a gene that encodes foran enzyme used in the viral life cycle, and most typically in the caseof HBV, DNA polymerase. Recently, it has been demonstrated that theefficacy of a drug against HBV infection can be prolonged, augmented, orrestored by administering the compound in combination or alternationwith a second, and perhaps third, antiviral compound that induces adifferent mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution, or other parameterof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

[0065] The anti-hepatitis B viral activity of β-L-2′-dA, β-L-2′-dC,β-L-2′-dU, β-L-2′-dG, β-L-2′-dT, β-L-dI, or other β-L-2′-nucleosidesprovided herein, or the prodrugs, phosphates, or salts of thesecompounds, can be enhanced by administering two or more of thesenucleosides in combination or alternation. Alternatively, for example,one or more of β-L-2′-dA, β-L-2′-dC, β-L-2′-dU, β-L-2′-dG, β-L-2′-dT,β-L-dI, or other β-L-2′-nucleosides provided herein can be administeredin combination or alternation with 3TC, FTC, L-FMAU, DAPD, famciclovir,penciclovir, BMS-200475, bis pom PMEA (adefovir, dipivoxil); lobucavir,ganciclovir, or ribavarin.

[0066] In any of the embodiments described herein, if theβ-L-2′-nucleoside of the present invention is administered incombination or alternation with a second nucleoside or nonnucleosidereverse transcriptase inhibitor that is phosphorylated to an activeform, it is preferred that a second compound be phosphorylated by anenzyme that is different from that which phosphorylates the selectedβ-L-2′-nucleoside of the present invention in vivo. Examples of kinaseenzymes are thymidine kinase, cytosine kinase, guanosine kinase,adenosine kinase, deoxycytidine kinase, 5′-nucleotidase, anddeoxyguanosine kinase.

[0067] Preparation of the Active Compounds

[0068] The 2′-deoxy-β-L-erythro-pentofuranonucleoside derivatives of thepresent invention are known in the art and can be prepared according tothe method disclosed by Holy, Collect. Czech. Chem. Commun. (1972),37(12), 4072-87 and Mol. Phys. (1967), 3(4), 386-95.

[0069] A general process for obtainingβ-L-erythro-pentofuranonucleosides (β-L-dN) is shown in FIG. 1, usingL-ribose or L-xylose as a starting material.

[0070] Mono, di, and triphosphate derivatives of the active nucleosidescan be prepared as described according to published methods. Themonophosphate can be prepared according to the procedure of Imai et al.,J. Org. Chem., 34(6), 1547-1550 (June 1969). The diphosphate can beprepared according to the procedure of Davisson et al., J. Org. Chem.,52(9), 1794-1801 (1987). The triphosphate can be prepared according tothe procedure of Hoard et al., J. Am. Chem. Soc., 87(8), 1785-1788(1965).

[0071] Experimental Protocols

[0072] Melting points were determined in open capillary tubes on aGallenkamp MFB-595-010 M apparatus and are uncorrected. The UVabsorption spectra were recorded on an Uvikon 931 (KONTRON)spectrophotometer in ethanol. ¹H-NMR spectra were run at roomtemperature in DMSO-d₆ with a Bruker AC 250 or 400 spectrometer.Chemical shifts are given in ppm, DMSO-d₅ being set at 2.49 ppm asreference. Deuterium exchange, decoupling experiments or 2D-COSY wereperformed in order to confirm proton assignments. Signal multiplicitiesare represented by s (singlet), d (doublet), dd (doublet of doublets), t(triplet), q (quadruplet), br (broad), m (multiplet). All J-values arein Hz. FAB mass spectra were recorded in the positive-(FAB>0) ornegative- (FAB<0) ion mode on a JEOL DX 300 mass spectrometer The matrixwas 3-nitrobenzyl alcohol (NBA) or a mixture (50:50, v/v) of glyceroland thioglycerol (GT). Specific rotations were measured on aPerkin-Elmer 241 spectropolarimeter (path length 1 cm) and are given inunits of 10⁻¹ deg cm² g⁻¹. Elemental analysis were carried out by the“Service de Microanalyses du CNRS, Division de Vernaison” (France).Analyses indicated by the symbols of the elements or functions werewithin ±0.4% of theoretical values. Thin layer chromatography wasperformed on precoated aluminium sheets of Silica Gel 60 F₂₅₄ (Merck,Art. 5554), visualisation of products being accomplished by UVabsorbency followed by charring with 10% ethanolic sulfuric acid andheating. Column chromatography was carried out on Silica Gel 60 (Merck,Art. 9385) at atmospheric pressure.

EXAMPLE 1

[0073] Stereospecific Synthesis of 2′-Deoxy-β-L-Adenosine

[0074] 9-(3,5-Di-O-benzoyl-β-L-xylofuranosyl)adenine (3)

[0075] A solution of9-(2-O-acetyl-3,5-di-O-benzoyl-β-L-xylofuranosyl)adenine 2 [Ref.:Gosselin, G.; Bergogne, M. -C.; Imbach, J. -L., “Synthesis and AntiviralEvaluation of β-L-Xylofuranosyl Nucleosides of the Five NaturallyOccuring Nucleic Acid Bases”, Journal of Heterocyclic Chemistry, 1993,30 (October-November), 1229-1233] (8.30 g, 16.05 mmol) and hydrazinehydrate 98% (234 mL, 48.5 inmol) in a mixture of pyridine/glacial aceticacid (4/1, v/v, 170 mL) was stirred at room temperature for 22 h. Thereaction was quenched by adding acetone (40 mL) and stirring wascontinued for one additional hour. The reaction mixture was reduced toone half of its volume, diluted with water (250 mL) and extracted withchloroform (2×150 mL). The organic layer was washed successively with anaqueous saturated solution of NaHCO₃ (3×100 mL) and water (3×100 mL),dried, filtered, concentrated and co-evaporated with toluene andmethanol. The residue was purified by silica gel column chromatography(0-3% MeOH in dichloromethane) to give 3 (5.2 g, 68%) precipitated fromdiisopropylic ether: ¹H NMR (DMSO-d₆): δ4.5-4.9 (m, 4H, H-2′, H-4′, H-5′and H-5″), 5.64 (t, 1H, H-3′, J_(2′,3′)=J_(3′,4′)=3.5 Hz), 6.3 (br s,1H, OH-2′), 6.45 (d, 1H, H-1′, J_(1′,2′)=4.6 Hz), 7.3 (br s, 2H, NH₂-6),7.4-7.0 (m, 10H, 2 benzoyls), 8.07 and 8.34 (2s, 2H, H-2 and H-8); ms:matrix G/T, (FAB⁺) m/z 476 [M+H]⁺, 136 [BH₂]⁺, (FAB⁻) m/z 474 [M−H]⁻,134 [B]⁻; UV (95% ethanol): λ_(max) 257 nm (ε16400), 230 nm (ε29300),λ_(max) 246 nm (ε14800); [α]_(D) ²⁰=−64 (c 1.07, CHCl₃). Anal. Calcd forC₂₄H₂₁N₅O₄ (M=475.45): C, 60.43; H, 4.45; N, 14.73. Found: C, 60.41; H,4.68; N, 14.27.

[0076] 9-(3,5-Di-O-benzoyl-2-deoxy-β-L-threo-pentofuranosyl)adenine (4).

[0077] To a solution of compound 3 (1.00 g, 2.11 mmol) in dryacetonitrile (65 mL) were added 4-(dimethylamino)pyridine (0.77 g, 6.32mmol) and phenoxythiocarbonyl chloride (0.44 mL, 3.16 mmol). The mixturewas stirred at room temperature for 2 h. After concentration, theresidue was dissolved in dichloromethane (50 mL) and washed successivelywith water (2×30 mL), aqueous solution of hydrochloric acid 0.5 N (30mL) and water (3×30 mL). The organic layer was dried, filtered andconcentrated to dryness. The crude thiocarbonylated intermediate wasdirectly treated with tris-(trimethylsilyl)silane hydride (0.78 mL, 5.23mmol) and α,α′-azoisobutyronitrile (AIBN, 0.112 g, 0.69 mmol) in drydioxane (17 mL) at reflux for 2 h. The solvent was removed under vacuumand the residue was purified by silica gel column chromatography (0-5%MeOH in dichloromethane) to give pure 4 (0.93 g, 96%) as a foam: ¹H NMR(DMSO-d₆): δ2.9-3.1 (m, 2H, H-2′ and H-2″), 4.6-4.7 (m, 3H, H-4′, H-5′and H-5″), 5.8 (br s, 1H, H-3′), 6.43 (dd, 1H, H-1′, J_(1′,2′)=3.1 Hz,J_(1′,2″)=7.6 Hz), 7.3 (br s, 2H, NH₂-6), 7.4-7.9 (m, 10H, 2 benzoyls),8.05 and 8.33 (2s, 2H, H-2 and H-8); ms: matrix G/T, (FAB⁺) m/z 460[M+H]⁺, 325 [S]⁺, 136 [BH₂]⁺, (FAB⁻) m/z 458 [M−H]⁻, 134 [B]⁻; UV (95%ethanol): λ_(max) 261 nm (ε14400), 231 nm (ε26300), λ_(min) 249 nm(ε12000); [α]_(D) ²⁰=−38 (c 1.04, DMSO).

[0078]6-N-(4-Monomethoxytrityl)-9-(3,5-di-O-benzoyl-2-deoxy-β-L-threo-pento-furanosyl)adenine(5).

[0079] To a solution of compound 4 (0.88 g, 1.92 mmol) in dry pyridine(40 mL) was added 4-monomethoxytrityl chloride (1.18 g, 3.84 mmol). Themixture was stirred at 60° C. for 24 h. After addition of methanol (5mL), the solution was concentrated to dryness, the residue was dissolvedin dichloromethane (50 mL) and washed successively with water (30 mL),aqueous saturated NaHCO₃ (30 mL) and water (30 mL). The organic layerwas dried, filtered, concentrated and co-evaporated with toluene to givepure 5 (1.01 g, 72%) as a foam: ¹H NMR (CDCl₃): δ2.9-3.0 (m, 2H, H-2′and H-2″), 3.62 (s, 3H, OCH₃), 4.6-4.8 (m, 3H, H-4′, H-5′ and H5″), 5.85(pt, 1H, H-3′), 6.44 (dd, 1H, H-1′, J_(1′,2′)=3.1 Hz, J_(1′,2″)=7.3 Hz),6.9 (br s, 1H, NH-6), 6.7-6.8 and 7.2-7.4 (2m, 24H, 2 benzoyls andMMTr), 7.97 and 8.13 (2s, 2H, H-2 and H-8); ms: matrix G/T, (FAB⁺) m/z732 [M+H]⁺, (FAB⁻) m/z 730 [M−H]⁻; UV (95% ethanol): λ_(max) 274 nm(ε12100), 225 nm (ε24200), λ_(min) 250 nm (ε5900); [α]_(D) ²⁰=−16 (c1.12, DMSO).

[0080]6-N-(4-Monomethoxytrityl)-9-(2-deoxy-β-L-threo-pentofuranosyl)-adenine(6).

[0081] Compound 5 (0.95 g, 1.30 mmol) was treated with a solution(saturated at −10° C.) of methanolic ammonia (40 mL), at roomtemperature overnight. After concentration, the residue was dissolved indichloromethane (60 mL) and washed with water (30 mL). The aqueous layerwas extracted twice with dichloromethane (10 mL). The combined organiclayer was dried, filtered and concentrated. The residue was purified bysilica gel column chromatography (0-5% MeOH in dichloromethane) to givepure 6 (0.67 g, 98%) as a foam: ¹H NMR (CDCl₃): δ2.6-2.9 (m, 2H, H-2′and H-2″), 3.5 (br s, 1H, OH-5′), 3.55 (s, 3H, OCH₃), 3.9-4.0 (m, 3H,H-4′, H-5′ and H-5″), 4.5-4.6 (m, 1H, H-3′), 6.03 (dd, 1H, H-1′,J_(1′,2′)=4.0 Hz, J_(1′,2″)=8.8 Hz), 7.0 (br s, 1H, NH-6), 6.7-6.8 and7.1-7.4 (2m, 14H, MMTr), 7.40 (d, 1H, OH-3′, J_(H,OH) =10.6 Hz), 7.80and 7.99 (2s, 2H, H-2 and H-8); ms: matrix G/T, (FAB⁺) m/z 524 [M+H]⁺,408 [BH₂]⁺, (FAB⁻), m/z 1045 [2M−H]⁻, 522 [M−H]⁻, 406 [B]⁻; UV (95%ethanol): λ_(max) 275 nm (ε12300), λ_(min) 247 nm (ε3600); [α]_(D)²⁰=+28 (c 0.94, DMSO).

[0082]6-N-(4-Monomethoxytrityl)-9-(2-deoxy-5-O-(4-monomethoxytrityl)-β-L-threo-pentofuranosyl)adenine(7).

[0083] Compound 6 (0.62 g, 1.24 mmol) in dry pyridine (25 mL) wastreated with 4-monomethoxytrityl chloride (0.46 g, 1.49 mmol) at roomtemperature for 16 h. After addition of methanol (5 mL), the mixture wasconcentrated to dryness. The residue was dissolved in dichloromethane(60 mL) and washed successively with water (40 mL), a saturated aqueoussolution of NaHCO₃ (40 mL) and water (3×40 mL). The organic layer wasdried, filtered, concentrated and co-evaporated with toluene andmethanol. The residue was purified by silica gel column chromatography(0-10% MeOH in dichloromethane) to give 7 (0.71 g, 72%) as a foam: ¹HNMR (DMSO-d₆): δ2.21 (d, 1H, H-2′ J_(2′,2″)=14.3 Hz), 2.6-2.7 (m, 1H,H-2″), 3.1-3.3 (2m, 2H, H-5′ and H-5″), 3.64 and 3.65 (2s, 6H, 2×OCH₃),4.1-4.2 (m, 1H, H-4′), 4.2-4.3 (m, 1H, H-3′), 5.68 (d, 1H, OH-3′,J_(H,OH)=5.2 Hz), 6.24 (d, 1H, H-1′, J_(1′,2″)=7.0 Hz), 6.7-6.8 and7.1-7.3 (2m, 29H, 2 MMTr and NH-6), 7.83 and 8.21 (2s, 2H, H-2 and H-8);m: matrix G/T, (FAB⁺) m/z 796 [M+H]⁺, 408 [BH₂]⁺, (FAB⁻) m/z 794 [M−H]⁻,406 [B]⁻; UV (95% ethanol): λ_(max) 275 nm (ε30900), λ_(min) 246 nm(ε12800); [α]_(D) ²⁰=+14 (c 1.03, DMSO).

[0084]6-N-(4-Monomethoxytrityl)-9-(3-O-benzoyl-2-deoxy-5-O-(4-mono-methoxytrityl)-β-L-erythro-pentofuranosyl)adenine(8).

[0085] A solution of diethylazodicarboxylate (0.38 mL, 2.49 mmol) in drytetrahydrofuran (20 mL) was added dropwise to a cooled solution (0° C.)of nucleoside 7 (0.66 g, 0.83 mmol), triphenylphosphine (0.66 g, 2.49mmol) and benzoic acid (0.30 g, 2.49 mmol) in dry THF (20 mL). Themixture was stirred at room temperature for 18 h and methanol (1 mL) wasadded. The solvents were removed under reduced pressure and the crudematerial was purified by silica gel column chromatography (0-5% ethylacetate in dichloromethane) to give compound 8 slightly contaminated bytriphenylphosphine oxide.

[0086]6-N-(4-Monomethoxytrityl)-9-(2-deoxy-5-O-(4-monomethoxytrityl)-β-L-erythro-pentofuranosyl)adenine(9).

[0087] Compound 8 was treated by a solution (saturated at −10° C.) ofmethanolic ammonia (20 mL), at room temperature for 24 h, then thereaction mixture was concentrated to dryness. The residue was dissolvedin dichloromethane (30 mL) and washed with water (20 mL). The aqueouslayer was extracted by dichloromethane (2×20 mL) and the combinedorganic phase was dried, filtered and concentrated. Pure compound 9(0.50 g, 76% from 7) was obtained as a foam after purification by silicagel column chromatography (0-2% MeOH in dichloromethane): ¹H NMR(DMSO-d₆): δ2.2-2.3 (m, 1H, H-2′), 2.8-2.9 (m, 1H, H-2″), 3.1-3.2 (m,2H, H-5′ and H-5″), 3.64 and 3.65 (2s, 6H, 2×OCH₃), 3.97 (pq, 1H, H-4′),4.4-4.5 (m, 1H, H-3′), 5.36 (d, 1H, OH-3′, J_(H,OH)=4.5 Hz), 6.34 (t,1H, H-1′, J_(1′,2′)=J_(1′,2″)=6.4 Hz), 6.8-6.9 and 7.1-7.4 (2m, 29H, 2MMTr and NH-6), 7.81 and 8.32 (2s, 2H, H-2 and H-8); ms: matrix G/T,(FAB⁺) m/z 796 [M+H]⁺, 408 [BH₂]⁺, (FAB⁻) m/z 794 [M−H]⁻, 406 [B]⁻; UV(95% ethanol): λ_(max) 276 nm (ε42600), λ_(min) 248 nm (ε23300); [α]_(D)²⁰=+29 (c 1.05, DMSO).

[0088] 2′-Deoxy-β-L-adenosine (β-L-dA)

[0089] Compound 9 (0.44 g, 0.56 mmol) was treated with an aqueoussolution of acetic acid 80% (17 mL) at room temperature for 5 h. Themixture was concentrated to dryness, the residue was dissolved in water(20 mL) and washed with diethyl ether (2×15 mL). The aqueous layer wasconcentrated and co-evaporated with toluene and methanol. The desired2′-deoxy-β-L-adenosine (β-L-dA) (0.12 g, 83%) was obtained afterpurification by silica gel column chromatography (0-12% MeOH indichloromethane) and filtration through a Millex HV-4 unit (0.45 μ,Millipore): mp 193-194° C. (crystallized from water) (Lit. 184-185° C.for L-enantiomer [Ref.: Robins, M. J.; Khwaja, T. A.; Robins, R. K. J.Org. Chem. 1970, 35, 636-639] and 187-189° C. for D-enantiomer [Ref.:Ness, R. K. in Synthetic Procedures in Nucleic Acid Chemistry; Zorbach,W. W., Tipson, R. S., Eds.; J. Wiley and sons: New York, 1968; Vol 1, PP183-187]; ¹H NMR (DMSO-d₆): δ2.2-2.3 and 2.6-2.7 (2m, 2H, H-2′ andH-2″), 3.4-3.6 (2m, 2H, H-5′ and H-5″), 3.86 (pq, 1H, H-4′), 4.3-4.4 (m,1H, H-3′), 5.24 (t, 1H, OH-5′, J_(H,OH)=5.8 Hz), 5.30 (d, 1H, OH-3′,J_(H,OH)=4.0Hz), 6.32 (dd, 1H, H-1′, J_(1′,2′)=6.2Hz, J_(1′,2″)=7.8 Hz),7.3 (br s, 2H, NH₂-6), 8.11 and 8.32 (2s, 2H, H-2 and H-8); ms: matrixG/T, (FAB⁺) m/z 252 [M+H]⁺, 136 [BH₂]⁺, (FAB⁻) m/z 250 [M−H]⁻, 134 [B]⁻;UV (95% ethanol): λ_(max) 258 nm (ε14300), λ_(min) 226 nm (ε2100);[α]_(D) ²⁰=+25 (c 1.03, H₂O), (Lit. [α]_(D) ²⁰=+23 (c 1.0, H₂O) forL-enantiomer [Ref.: Robins, M. J.; Khwaja, T. A.; Robins, R. K. J. Org.Chem. 1970, 35, 636-639] and [α]_(D) ²⁰=−25 (c 0.47, H₂O) forD-enantiomer [Ref.: Ness, R. K. in Synthetic Procedures in Nucleic AcidChemistry; Zorbach, W. W., Tipson, R. S., Eds.; J. Wiley and sons: NewYork, 1968; Vol 1, PP 183-187]). Anal. Calcd for C₁₀H₁₃N₅O₃+1.5 H₂O(M=278.28): C, 43.16; H, 5.80; N, 25.17. Found: C, 43.63; H, 5.45; N,25.33.

EXAMPLE 2

[0090] Stereoselective Synthesis of 2′-Deoxy-β-L-Adenosine (β-L-dA)

[0091] Reaction 1:

[0092] Precursor: L-ribose (Cultor Science Food, CAS [24259-59-4], batchRIB9711013)

[0093] Reactants: Sulphuric acid 95-97% (Merck; ref 1.00731.1000);Benzoyl chloride (Fluka; ref 12930); Sodium sulfate (Prolabo; ref28111.365)

[0094] Solvents: Methanol P.A. (Prolabo; ref 20847.295); Pyridine 99%(Acros; ref 131780025); Dichloromethane P.A. (Merck; ref 1.06050.6025);Acetic acid P.A. (carlo erba; ref 20104298); Acetic anhydride (Fluka;ref 45830); Ethanol 95 (Prolabo; ref 20823.293)

[0095] References: Recondo, E. F., and Rinderknecht, H., Eine neue,Einfache Synthese des 1-O-Acetyl-2,3,5-Tri-O-β-D-Ribofuranosides. Helv.Chim. Acta, 1171-1173 (1959).

[0096] A solution of L-ribose 140 (150 g, 1 mol) in methanol (2 liters)was treated with sulphuric acid (12 ml) and left at +4° C. for 12 hrs,and then neutralised with pyridine (180 ml). Evaporation gave an α,βmixture of methyl ribofuranosides 141 as a syrup. A solution of thisanomeric mixture in pyridine (1.3 liters) was treated with benzoylchloride (580 ml, 5 mol) with cooling and mechanical stirring. Thesolution was left at room temperature for 12 hrs and then poured on ice(about 10 liters) with continued stirring. The mixture (an oil in water)was filtered on a Cellite bed. The resulting oil on the cellite bed waswashed with water (3×3 liters) and then dissolved with ethyl acetate (3liters). The organic phase was washed with a 5% NaHCO₃ solution (2liters) and water (2 liters), dried over sodium sulfate, filtered andevaporated to give 1-O-methyl-2,3,5-tri-O-benzoyl-α/β-L-ribofuranose 142as a thick syrup. The oil was dissolved in acetic anhydride (560 ml) andacetic acid (240 ml). The solution was, after the dropwise addition ofconcentrated sulphuric acid (80 ml), kept in the cold (+4° C.) undermechanical stirring for 10 hrs. The solution was then poured on ice(about 10 liters) under continued stirring. The mixture (oily compoundin water) was filtered on a Cellite bed. The resulting gummy solid onthe cellite bed was washed with water (3×3 liters) and then dissolved indichloromethane (2,5 liters). The organic phase was washed with 5%NaHCO₃ (1 liter) and water (2×2 liters), dried over sodium sulfate,filtered and evaporated to give a gummy solid 143, which wascrystallized from ethanol 95 (yield 225 g, 44%).

[0097] Analyses for 1-O-acetyl-2,3,5-tri-O-benzoyl-β-L-ribofuranose 143:

[0098] mp 129-130° C. (EtOH 95) (lit.(1) mp 130-131° C.)

[0099]¹H NMR (200 MHz, CDCl₃): δ8.09−7.87 (m, 6H, H_(Arom)), 7.62−7.31(m, 9H, H_(Arom)) 6.43 (s, 1H, H₁), 5.91 (dd, 1H, H₃, J_(3,4) 6.7 Hz;J_(3,2) 4.9 Hz), 5.79 (pd, 1H, H₂, J_(2,3) 4.9 Hz; J_(1,2)<1), 4.78 (m,2H, H₄ and H₅), 4.51 (dd, 1H, H₅, J_(5,5′)13.1 Hz, J_(5′,4) 5.5 Hz),2.00 (s, 3H, CH₃CO), (identical to commercial1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose)

[0100] Mass analysis (FAB+, GT) m/z 445 (M-OAc)⁺

[0101] Elemental analysis C₂₈H₂₄O₉ Calculated C 66.66 H 4.79; found CH

[0102] Reaction 2:

[0103] Precursor: Adenine (Pharma-Waldhof; ref 400134.001 lot 45276800)

[0104] Reactants: Stannic chloride fuming (Fluka; ref 96558);NH₃/Methanol (methanol saturated with NH₃; see page 5); Sodium sulfate(Prolabo; ref 28111.365)

[0105] Solvents: Acetonitrile (Riedel-de Hean; ref 33019; distilled overCaH₂); Chloroform Pur (Acros; ref 22706463); Ethyl acetate Pur (Carloerba; ref 528299)

[0106] References: Saneyoshi, M., and Satoh, E., Synthetic Nucleosidesand Nucleotides. XIII. Stannic Chloride Catalyzed Ribosylation ofSeveral 6-Substituted Purines. Chem; Pharm. Bull., 27, 2518-2521(1979).; Nakayama, C., and Saneyoshi, M., Synthetic Nucleosides andNucleotides. XX. Synthesis of Various 1-β-Xylofuranosyl-5-Alkyluracilsand Related Nucleosides. Nucleosides, Nucleotides, 1, 139-146 (1982).

[0107] Adenine (19.6 g, 144 mmol) was suspended in acetonitrile (400 ml)with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-L-ribofuranose 143 (60 g, 119mmol). To this suspension was added stannic chloride filming (22 ml, 187mmol). After 12 hrs, the reaction was concentrated to a small volume(about 100 ml), and sodium hydrogencarbonate (110 g) and water (120 ml)were added. The resulting white solid (tin salts) was extracted with hotchloroform (5×200 ml). The combined extracts were filtered on a cellitebed. The organic phase was washed with a NaHCO₃ 5% solution and water,dried over sodium sulfate, filtered and evaporated to give compound 144(60 g, colorless foam). The foam was treated with methanol saturatedwith ammonia (220 ml) in sealed vessel at room temperature understirring for 4 days. The solvent was evaporated off under reducedpressure and the resulting powder was suspended in ethyl acetate (400ml) at reflux for 1 hr. After filtration, the powder was recrystallizedfrom water (220 ml) to give L-adenosine 145 (24 g, crystals, 75%)

[0108] Analyses for β-L-adenosine:

[0109] mp 233-234° C. (water) (lit.(4) mp 235°-238° C.)

[0110]¹H NMR (200 MHz, DMSO-D₆): δ8.34 and 8.12 (2s, 2H, H₂ and H₈),7.37 (1s, 2H, NH₂), 5.86 (d, 1H, H_(1′), J_(1′2′)6.2 Hz), 5.43 (m, 2H,OH_(2′) and OH_(5′)), 5.19 (d, 1H, OH_(3′), J 3.7 Hz), 4.60 (m, H_(2′)),4.13 (m, 1H, H_(3′)), 3.94 (m, 1H, H_(4′)) 3.69−3.49 (m, 2H, H_(5′a) andH_(5′b)), (identical to commercial D-adenosine)

[0111] Mass analysis (FAB+, GT) m/z 268 (M+H)⁺, 136(BH2)⁺

[0112] Reaction 3:

[0113] Reactants: 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (Fluka;ref 36520); Sodium sulfate (Prolabo; ref 28111.365)

[0114] Solvents: Pyridine 99% (Acros; ref 131780025); Ethyl acetate Pur(Carlo erba; ref 528299); Acetonitrile (Riedel-de Haen; ref 33019)

[0115] Reference: Robins, M. J., et al., Nucleic Acid Related Compounds.42. A General Procedure for the Efficient Deoxygenation of SecondaryAlcohols. Regiospecific and Stereoselective Conversion ofRibonucleosides to 2′-Deoxynucleosides. J. Am. Chem. Soc. 105, 4059-4065(1983).

[0116] To L-adenosine 145 (47.2 g, 177 mmol) suspended in pyridine (320ml) was added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (63 ml, 201mmol), and the mixture was stirred at room temperature for 12 hrs.Pyridine was evaporated and the residue was partitioned with ethylacetate (1 liter) and a NaHCO₃ 5 % solution (600 ml). The organic phasewas washed with a HCl 0.5N solution (2×500 ml) and water (500 ml), driedover sodium sulfate, filtered and evaporated to dryness. The resultingsolid was crystallized from acetonitrile to give compound 146 (81 g,90%).

[0117] Analyses3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-β-L-adenosine 146:

[0118] mp 97-98° C. (acetonitrile) (lit. (5) D enantiomer mp 98° C.)

[0119]¹H NMR (200 MHz, CDCl₃): δ8.28 and 7.95 (2s, 2H, H₂ and H₈), 5.96(d, 1H, J_(1′,2′)1.1 Hz), 5.63 (s, 2H, NH₂), 5.10 (dd, 1H, H_(3′),J_(3′,4′)7.6 Hz, J_(3′,2′)5.5 Hz), 4.57 (dd, 1H, H_(2′), J_(2′,1′)1.2Hz; J_(2′,3′)7.6 Hz), 4.15−3.99 (m, 3H, H_(4′), H_(5′a) and H_(5′b)),3.31 (s1, 1H, OH_(2′)), 1.06 (m, 28H, isopropyl protons)

[0120] Mass analysis (FAB−, GT) m/z 508 (M−H)⁻, 134 (B)⁻; (FAB+, GT) m/z510 (m+H)⁺, 136 (BH₂)⁺

[0121] Reaction 4:

[0122] Reactants: Dimethylaminopyridine 99% (Acros; ref 1482702050);Phenylchlorothionocarbonate 99% (Acros; ref 215490050);Tris(trimethylsilyl)silane “TTMSS” (Fluka; ref 93411);α,α′-Azoisobutyronitrile “AIBN” (Fluka, ref 11630); Sodium sulfate(Prolabo; ref 28111.365)

[0123] Solvents: Acetonitrile (Riedel-de Haen; ref 33019); Ethyl acetatePur (Carlo Erba; ref 528299); Dioxan P.A. (Merck; ref 1.09671.1000);Dichloromethane (Merck; ref 1.06050.6025); Methanol (Carlo Erba; ref309002);

[0124] Reference: Robins, M. J., Wilson, J. S., and Hansske, F., NucleicAcid Related Compounds. 42. A General Procedure for the EfficientDeoxygenation of Secondary Alcohols. Regiospecific and StereoselectiveConversion of Ribonucleosides to 2′-Deoxynucleosides. J. Am. Chem. Soc.,105, 4059-4065 (1983).

[0125] To compound 146 (34 g, 67 mmol) were added acetonitrile (280 ml),DMAP (16.5 g, 135 mmol) and phenyl chlorothionocarbonate (10.2 ml, 73mmol). The solution was stirred at room temperature for 12 hrs. Solventwas evaporated and the residue was partioned between ethyl acetate (400ml) and a HCl 0.5N solution (400 ml). The organic layer was washed witha HCl 0.5N solution (400 ml) and water (2×400 ml), dried over sodiumsulfate, filtered and evaporated to dryness to give the intermediate asa pale yellow solid. The crude 147 was dissolved in dioxan (ml) and AIBN(3.3 g, 20 mmol) and TTMSS (33 ml, 107 mmol) were added. The solutionwas progressively heated until reflux and stirred for 2 hrs. Thereaction was concentrated to a yellow oil which was chromatographed(eluent dichloromethane/methanol 95/5) to give compound 148 (23 g,colorless foam, 70%). An aliquot was cristallized from ethanol/petroleum ether.

[0126] Analyses for3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-2′-deoxy-β-L-adenosine148:

[0127] mp 110-111° C. (EtOH/petroleum ether) (Lit.(5) mp 113-114° C.(EtOH))

[0128]¹H NMR (200 MHz, CDCl₃): δ8.33 and 8.03 (2s, 2H, H₂ and H₈), 6.30(dd, 1H, H_(1′), J 2.85 Hz, J 7.06 Hz), 5.63 (s1, 2H, NH₂), 4.96 (m, 1H,H_(3′)), 4.50 (m, 2H, H_(5′a) and H_(5′b)), 2.68 (m, 2H, H_(2′a) andH_(2′b)), 1.08 (m, 28H, isopropyl protons)

[0129] Mass analysis (FAB+, GT) m/z 494 (M+H)⁺, 136 (BH₂)⁺

[0130] Reaction 5:

[0131] Reactants: Ammonium fluoride (Fluka; ref 09742); Silica gel(Merck; ref 1.07734.2500)

[0132] Solvents: Methanol P.A. (Prolabo; ref 20847.295); DichloromethaneP.A. (Merck; ref 1.06050.6025); Ethanol 95 (Prolabo; ref 20823.293)

[0133] Reference: Zhang, W., and Robins, M. J., Removal of SilylProtecting Groups from Hydroxyl Functions with Ammonium Fluoride inMethanol. Tetrahedron Lett., 33, 1177-1180 (192).

[0134] A solution of3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-2′-deoxy-L-adenosine148 (32 g, 65 mmol) and ammonium fluoride (32 g, mmol) in methanol wasstirred at reflux for 2 hrs. Silica gel was added and the mixture wascarefully evaporated to give a white powder. This powder was added onthe tpo of a silica column, which was eluted withdichloromethane/methanol 9/1. The appropriate fractions were combinedand evaporated to give a white powder, which was crystallized fromethanol 95 (12.1 g, 75%).

[0135] Analyses for 2′-Deoxy-β-L-adenosine 149:

[0136] mp 189-190° C. (EtOH 95) (identical to commercial2′-deoxy-D-adenosine)

[0137]¹H NMR (200 MHz, DMSO-D₆): δ8.35 and 8.14 (2s, 2H, H₂ and H₈),7.34 (s1, 2H, NH₂), 6.35 (dd, 1H, H1′, J 6.1 Hz, J 7.85 Hz), 5.33 (d,1H, OH_(2′), J 4.0 Hz), 5.28 (dd, 1H, H_(3′), J 4.9 Hz; J 6.6 Hz), 4.42(m, 1H, OH_(5′)), 3.88 (m, 1H, H_(4′)), 3.63−3.52 (m, 2H, H_(5′a) andH_(5′b)), 2.71 (m, 1H, H_(2′a)), 2.28 (m, 1H, H_(2′b)). (identical tocommercial 2′-deoxy-D-adenosine)

[0138] α_(D)+26° (c 0.5 water) (commercial 2′-deoxy-D-adenosine −25° (c0.5 water)).

[0139] UV λ_(max) 260 nm (ε14100) (H₂O).

[0140] Mass analysis (FAB+, GT) m/z 252 (M+H)⁺, 136 (BH₂)⁺

EXAMPLE 3

[0141] Stereospecific Synthesis of 2′-Deoxy-β-L-Cytidine

[0142] 1-(3,5-Di-O-benzoyl-β-L-xylofuranosyl)uracil (11)

[0143] Hydrazine hydrate (1.4 mL, 28.7 mmol) was added to a solution of1-(2-O-acetyl-3,5-di-O-benzoyl-β-L-xylofuranosyl)uracil 10 [Ref.:Gosselin, G.; Bergogne, M. -C.; Imbach, J. -L., “Synthesis and AntiviralEvaluation of β-L-Xylofuranosyl Nucleosides of the Five NaturallyOccuring Nucleic Acid Bases”, Journal of Heterocyclic Chemistry, 1993,30 (October-November), 1229-1233] (4.79 g, 9.68 mmol) in pyridine (60mL) and acetic acid (15 mL). The solution was stirred overnight at roomtemperature. Acetone was added (35 mL) and the mixture was stirred for30 min. The reaction mixture was evaporated under reduced pressure. Theresulting residue was purified by silica gel column chromatography[eluent: stepwise gradient of methanol (0-4%) in dichloromethane to give11 (3.0 g, 68%) which was crystallized from cyclohexane/dichloromethane:mp=111-114° C.; ¹H-NMR (DMSO-d₆): δ11.35 (br s, 1H, NH), 7.9−7.4 (m,11H, 2 C₆H₅CO, H-6), 6.38 (d, 1H, OH-2′, J_(OH-2′)=4.2 Hz), 5.77 (d, 1H,H-1′, J_(1′,2′)=1.9 Hz), 5.55 (d, 1H, H-5, J₅₋₆=8 Hz), 5.54 (dd, 1H,H-3′, J_(3′-2′)=3.9 Hz and J_(3′-4′)=1.8 Hz), 4.8 (m, 1H, H-4′), 4.7 (m,2H, H-5′ and H-5″), 4.3 (m, 1H, H-2′); MS: FAB>0 (matrix GT) m/z 453(M+H)⁺, 105 (C₆H₅CO)⁺; FAB<0 (matrix GT) m/z 451 (M−H)⁻, 121 (C₆H₅CO₂)⁻,111 (B)⁻; Anal. Calcd for C₂₃H₂₀N₂O₈.H₂O: C, 58.09 ; H, 4.76 ; N, 5.96.Found: C, 57.71; H, 4.42; N, 5.70.

[0144] 1-(3,5-Di-O-benzoyl-β-L-arabinofuranosyl)uracil (12)

[0145] To a solution of 1-(3,5-di-O-benzoyl-β-L-xylofuranosyl)uracil 11(8 g, 17.7 mL) in an anhydrous benzene-DMSO mixture (265 mL, 6:4, v/v)were added anhydrous pyridine (1.4 mL), dicyclohexylcarbodiimide (10.9g, 53 mmol) and dichloroacetic acid (0.75 mL). The resulting mixture wasstirred at room temperature for 4 h, then diluted with ethyl acetate(400 mL) and a solution of oxalic acid (4.8 g, 53 mmol) in methanol (14mL) was added. After being stirred for 1 h, the solution was filtered.The filtrate was washed with a saturated NaCl solution (2×500 mL), 3%NaHCO₃ solution (2×500 mL) and water (2×500 mL). The organic phase wasdried over Na₂SO₄, then evaporated under reduced pressure. The resultingresidue was then solubilized in an EtOH absolute-benzene mixture (140mL, 2:1, v/v). To this solution at 0° C. was added NaBH₄ (0.96 g, 26.5mmol). After being stirred for 1 h, the solution was diluted with ethylacetate (400 mL), then filtered. The filtrate was washed with asaturated NaCl solution (400 mL) and water (400 mL). The organic phasewas dried over Na₂SO₄, then evaporated under reduced pressure. Theresulting crude material was purified by silica gel columnchromatography [eluent: stepwise gradient of methanol (0-3%) indichloromethane to give 12 (5.3 g, 66%) which was crystallized fromacetontrile: mp=182-183° C.; ¹H-NMR (DMSO-d₆): δ11.35 (br s, 1H, NH),8.0−7.5 (m, 11H, 2 C₆H₅CO, H-6), 6.23 (br s, 1H, OH-2′), 6.15 (d, 1H,H-1′, J_(1′-2′)=4 Hz), 5.54 (d, 1H, H-5, J₅₋₆=8.1 Hz), 5.37 (t, 1H,H-3′, J_(3′-2′)=J_(3′-4′)=2.6 Hz), 4.7−4.6 (m, 2H, H-5′ and H-5″), 4.5(m, 1H, H-4′), 4.4 (m, 1H, H-2′); MS: FAB>0 (matrix GT) m/z 453 (M+H)⁺,341 (S)⁺, 113 (BH₂)⁺, 105 (C₆H₅CO)⁺; FAB<0 (matrix GT) m/z 451 (M−H)⁻,121 (C₆H₅CO₂)⁻, 111 (B)⁻; Anal. Calcd for C₂₃H₂₀N₂O₈: C, 61.06 ; H, 4.46; N, 6.19. Found: C, 60.83; H, 4.34; N, 6.25.

[0146] 1-(3,5-Di-O-benzoyl-2-deoxy-β-L-erythro-pentofuranosyl)uracil(13)

[0147] To a solution of 1-(3,5-di-O-benzoyl-β-L-arabinofuranosyl)uracil12 (5.2 g, 11.4 mmoL) in anhydrous 1,2-dichloroethane (120 mL) wereadded phenoxythiocarbonyl chloride (4.7 mL, 34.3 mL) and4-(dimethylamino)pyridine (DMAP, 12.5 g, 102.6 mmoL). The resultingsolution was stirred at room temperature under argon atmosphere for 1 hand then evaporated under reduced pressure. The residue was dissolved indichloromethane (300 mL) and the organic solution was successivelywashed with an ice-cold 0.2 N hydrochloric acid solution (3×200 mL) andwater (2×200 mL), dried over Na₂ SO₄ then evaporated under reducedpressure. The crude material was co-evaporated several times withanhydrous dioxane and dissolved in this solvent (110 mL). To theresulting solution were added under argon tris-(trimethylsilyl)silanehydride (4.2 mL, 13.7 mmol) and α,α′-azoisobutyronitrile (AIBN, 0.6 g,3.76 mmol). The reaction mixture was heated and stirred at 100° C. for 1h under argon, then cooled to room temperature and evaporated underreduced pressure. The residue was purified by silica gel columnchromatography [eluent: stepwise gradient of methanol (0-5%)] to give 13(2.78 g, 56%) which was crystallized from EtOH: mp=223-225° C.; ¹H-NMR(DMSO-d₆): δ11.4 (br s, 1H, NH), 8.0−7.5 (m, 11H, 2 C₆H₅CO, H-6), 6.28(t, 1H, H-1′, J=7 Hz), 5.5 (m, 2H, H-1′ and H-5), 4.6−4.4 (m, 3H, H-4′,H-5′ and H-5″), 2.6 (in, 2H, H-2′ and H-2″); MS: FAB>0 (matrix GT) m/z437 (M+H)⁺, 3325 (s)⁺, FAB<0 (matrix GT) m/z 435 (M−H)⁻, 111 (B)⁻; Anal.Calcd for C₂₃H₂₀N₂O₇: C, 63.30; H, 4.62; N, 6.42. Found: C, 62.98;H14.79; N, 6.40.

[0148] 2′-Deoxy-β-L-cytidine (β-L-dC)

[0149] Lawesson's reagent (1.72 g, 4.26 mmol) was added under argon to asolution of1-(3,5-di-O-benzoyl-2-deoxy-β-L-erythro-pentofuranosyl)uracil 13 (2.66g, 6.1 mmol) in anhydrous 1,2-dichloroethane (120 mL) and the reactionmixture was stirred under reflux for 2 h. The solvent was thenevaporated under reduced pressure and the residue was purified by silicagel column chromatography [eluent: stepwise gradient of ethyl acetate(0-8%) in dichloromethane] to give the 4-thio intermediate as a yellowfoam. A solution of this thio-intermediate (1.5 g, 3.31 mmol) inmethanolic ammonia (previously saturated at −10° C. and tightly stopped)(50 mL) was heated at 100° C. in a stainless-steel bomb for 3 h and thencooled to 0° C. The solution was evaporated under reduced pressure. Theresulting crude material was purified by silica gel columnchromatography [eluent: stepwise gradient of methanol(0-20%) indichloromethane]. Finally, the appropriate fractions were pooled,filtered through a unit Millex HV-4 (0.45 μm, Millipore) and evaporatedunder reduced pressure to provide the desired 2′-deoxy-β-L-cytidine(β-L-dC) as a foam (0.6 g, 80%) which was crystallized from absoluteEtOH: mp=198-199° C.; ¹H-NMR (DMSO-d₆): δ7.77 (d, 1H, H-6, J₆₋₅=7.4 Hz),7.10 (br d, 2H, NH-₂), 6.13 (t, 1H, H-1′, J=6.7 Hz), 5.69 (d, 1H, H-5,J₅₋₆=7.4 Hz), 5.19 (d, 1H, OH-3′, J_(OH) _(⁻) _(3′)=4.1 Hz), 4.96 (t,1H, OH-5′, J_(OH-5′)=OH-_(5″)=5.2 Hz), 4.1 (m, 1H, H-3′), 3.75 (m, 1H,H-4′), 3.5 (m, 2H, H-5′ and H-5″), 2.0 (m, 1H, H-2′), 1.9 (m, 1H, H-2″);MS: FAB>0 (matrix GT) m/z 228 (M+H)⁺, 112 (BH₂)⁺; FAB<0 (matrix GT) m/z226(M−H)⁻; [α]²⁰ _(D)=−69 (c 0.52, DMSO) [[α]²⁰ _(D)=+76 (c 0.55, DMSO)for a commercially available hydrochloride salt of the D-enantiomer].Anal. Calcd for C₉H₁₃N₃O₄: C, 47.57; H, 5.77; N, 18.49. Found: C, 47.35;H, 5.68; N, 18.29.

EXAMPLE 4

[0150] Stereoselective Synthesis of 2′-Deoxy-β-L-Cytidine (β-L-dC)

[0151] 2-Amino-β-L-arabinofurano[1,2′:4,5]oxazoline (1)

[0152] A mixture of L-arabinose (170 g, 1.13 mol), cyanamide (100 g,2.38 mol), methanol (300 ml), and 6M-NH₄OH (50 ml) was stirred at roomtemperature for 3 days and then kept at −10° C. overnight. The productwas collected with suction, washed successively with methanol and ether,and dried in vacuo. Yield, 130 g (66.0%) of the analytically purecompound 1, m.p. 170-172° C.;

[0153]¹H NMR (DMSO-d₆) δppm 6.35 (br s, 2H, NH₂), 5.15 (d, 1H, H-1,J=5.6 Hz), 5.45 (br s, 1H), OH-3), 4.70 (br s, 1H, OH-5), 4.55 (d, 1H,H-2, J=5.6 Hz), 4.00 (br s, 1H, H-3), 3.65 (m, 1H, H-4), 3.25 (m, 2H,H-5, H-5′).

[0154] Reagents:

[0155] L-arabinose: Fluka, >99.5%, ref 10839

[0156] Cyanamide: Fluka, >98%, ref 28330

[0157] O^(2,2′)-anhydro-β-L-uridine (2)

[0158] A solution of compound 1 (98.8 g, 0.57 mol) and methyl propiolate(98 ml) in 50% aqueous ethanol (740 ml) was refluxed for 5 h, thencooled and concentrated under diminished pressure to the half of theoriginal volume. After precipitation with acetone (600 ml), the productwas collected with suction, washed with ethanol and ether, and dried.The mother liquor was partialy concentrated, the concentrateprecipitated with acetone (1000 ml), the solid collected with suction,and washed with acetone and ether to afford another crop of the product.Over-all yield, 80 g (62%) of compound 2, m.p. 236-240° C.; ¹H NMR(DMSO-d₆) δppm 7.87 (d, 1H, H-6, J=7.4 Hz), 6.35 (d, 1H, H-1′, J=5.7Hz), 5.95 (d, 1H, H-5, J=7.4 Hz), 5.90 (d, 1H, OH-3′), 5.20 (d, 1H,H-2′, J=5.7 Hz), 5.00 (m, 1H, OH-3′), 4.44 (br s, 1H, H-3′), 4.05 (m,1H, H-4′), 3.25 (m, 2H, H-5, H-5′)

[0159] Reagent:

[0160] Methyl propiolate: Fluka, >97%, ref 81863

[0161] 3′,5′-Di-O-benzoyl-O^(2,2′)-anhydro-β-L-uridine (3)

[0162] To a solution of compound 2 (71.1 g, 0.31 mol) in anhydrouspyridine (1200 ml) was added benzoyl chloride (80.4 ml) at 0° C. andunder argon. The reaction mixture was stirred at room temperature for 5h under exclusion of atmospheric moisture and stopped by addition ofethanol. The solvents were evaporated under reduced pressure and theresulting residue was coevaporated with toluene and absolute ethanol.The crude mixture was then diluted with ethanol and the precipitatecollected with suction, washed successively with ethanol and ether, anddried.

[0163] Yield, 129 g (95.8%) of compound 3, m.p. 254° C.; ¹H NMR(DMSO-d₆) δppm 8.1−7.4 (m, 11H, C₆H₅CO, H-6), 6.50 (d, 1H, H-1′, J=5.7Hz), 5.90 (d, 1H, H-5, J=7.5 Hz), 5.80 (d, 1H, H-2′, J=5.8 Hz), 5.70 (d,1H, H-3′) 4.90 (m, 1H, H-4′), 4.35 (m, 2H, H-5, H-5′).

[0164] Reagent:

[0165] Benzoyl chloride: Fluka, p.a., ref 12930

[0166] 3′,5′-Di-O-benzoyl-2′-chloro-2′-deoxy-β,L-uridine (4)

[0167] To a solution of compound 3 (60.3 g, 0.139 mol) indimethylformamide (460 ml) was added at 0° C. a 3.2 N-HCl/DMF solution(208 ml, prepared in situ by adding 47.2 ml of acetyl chloride at 0° C.to a solution of 27.3 ml of methanol and 133.5 ml of dimethylformamide).The reaction mixture was stirred at 100° C. for 1 h under exclusion ofatmospheric moisture, cooled down, and poured into water (4000 ml). Theprecipitate of compound 4 was collected with suction, washed with water,and recrystallised from ethanol. The crystals were collected, washedwith cold ethanol and ether, and dried under diminished pressure. Yield,60.6 g (92.6%) of compound 4, m.p. 164-165° C.; ¹H NMR (DMSO-d₆) δppm8.7 (br s, 1H, NH), 8.1−7.3 (m, 11H, C₆H₅CO, H-6), 6.15 (d, 1H, H-1′,J=4.8 Hz), 5.5 (m, 2H, H-5, H-2′), 4.65 (m, 4H, H-3′, H-4′, H-5′, H-5″).

[0168] Reagent:

[0169] Acetyl chloride: Fluka, p.a., ref 00990

[0170] 3′,5′-Di-O-benzoyl-2′-deoxy-β,L-uridine (5)

[0171] A mixture of compound 4 (60.28 g, 0.128 mol), tri-n-butyltinhydride (95 ml) and azabisisobutyronitrile (0.568 g) in dry toluene (720ml) was refluxed under stirring for 5 h and cooled down. The solid wascollected with suction and washed with cold toluene and petroleum ether.The filtrate was concentrated under reduced pressure and diluted withpetroleum ether to deposit an additional crop of compound 5. Yield,54.28 g (97.2%) of compound 5; m.p. 220-221° C.; ¹H NMR (CDCl₃) δppm8.91 (br s, 1H, NH), 8.1−7.5 (m, 11H, C₆H₅CO and H-6), 6.43 (q, 1H,H-1′, J_(1′,2′)=57 Hz and J_(1′,2″)=8.3 Hz), 5.7−5.6 (m, 2H, H-3′ andH-5), 4.8−4.6 (m, 3H, H-5′, H-5″ and H-4′), 2.8−2.7 (m, 1H, H-2′),2.4−2.3 (m, 1H, H-2″).

[0172] Reagents:

[0173] Tri-n-butyltin hydride: Fluka, >98%, ref 90915

[0174] Azabisisobutyronitrile: Fluka, >98%, ref 11630

[0175] 3′,5′-Di-O-benzoyl-2′-deoxy-β-L-4-thio-uridine (6)

[0176] A solution of compound 5 (69 g, 0.158 mol) and Lawesson's reagent(74 g) in anhydrous methylene chloride (3900 ml) was refluxed underargon overnight. After evaporation of the solvant, the crude residue waspurified by a silica gel column chromatography [eluant: gradient ofmethanol (0-2%) in methylene chloride] to afford pure compound 6 (73 g)in quantitative yield; ¹H NMR (CDCl₃) δppm 9.5 (br s, 1H, NH), 8.1−7.4(m, 10H, C₆H₅CO), 7.32 (d, 1H, H-6, J=7.7 Hz), 6.30 (dd, 1H, H-1′, J=5.6Hz and J=8.2 Hz), 6.22 (d, 1H, H-5, J=7.7 Hz), 5.6 (m, 1H, H-3′), 4.7(m, 2H, H-5′, H-5″), 4.5 (m, 1H, H-4′), 2.8 (m, 1H, H-2′), 2.3 (m, 1H,H-2″).

[0177] Reagent:

[0178] Lawesson's reagent: Fluka, >98%, ref 61750

[0179] 2′-Deoxy-β-L-cytosine

[0180] A solution of compound 6 (7.3 g, 0.016 mol) in methanol saturatedwith ammonia (73 ml) was heated at 100° C. in a stainless steel cylinderfor 3 h. After cooling carefully, the solvent was evaporated underreduced pressure. An aqueous solution of the residue was washed withethyl acetate and evaporated to dryness. Such a procedure was carriedout on 9 other samples (each 7.3 g) of compound 6 (total amount of 6=73g). The 10 residues were combined, diluted with absolute ethanol andcooled to give 7 as crystals. Trace of benzamide were eliminated fromthe crystals of 6 by a solid-liquid extraction procedure (at reflux inethyl acetate for 1 h). Yield, 28.75 g (78.6%) of compound 6; m- p.141-145° C.; ¹H NMR (DMSO) δppm 8.22 and 8.00 (2 br s, 2H, NH₂), 7.98(d, 1H, H-6, J=7.59 Hz), 6.12 (t, 1H, H-1′, J=6.5 Hz and J=7.6 Hz), 5.89(d, 1H, H-5, J=7.59 Hz), 5.3 (br s, 1H, OH-3′), 5.1 (br s, 1H, OH-5′),4.2 (m, 1H, H-3′), 3.80 (q, 1H, H-4′, J=3.6 Hz and J=6.9 Hz), 3.6−3.5(m, 2H, H-5′, H-5″), 2.2−2.0 (m, 2H, H-2′, H-2″); FAB<0, (GT) m/e 226(M−H)⁻, 110 (B)⁻; FAB>0 (GT) 228 (M+H)⁺, 112 (B+2H)⁺; [α]_(D) ²⁰−56.48(c=1.08 in DMSO); UV (pH 7) λ_(max)=270 nm (ε=10000).

[0181] Reagent:

[0182] Methanolic ammonia: previously saturated at −5° C., tightlystoppered, and kept in a freezer.

EXAMPLE 5

[0183] Stereoselective Synthesis of 2′-Deoxy-β-L-Thymidine (β-L-dT)

[0184] 3′,5′-Di-O-benzoyl-2′-deoxy-5-iodo-β-L-uridine (7)

[0185] A mixture of compound 5 (105.8 g, 0.242 mol), iodine (76.8 g),CAN (66.4 g) and acetonitrile (2550 ml) was stirred at 80° C. for 3 hthen the reaction mixture was cooled at room temperature leading tocrystallization of compond 7 (86.6 g, 63.5%); m.p. 192-194° C.; ¹H NMR(DMSO) δppm 0.8.34 (s, 1H, NH), 8.2−7.2 (m, 11H,2 C₆H₅CO, H-6), 6.31 (q,1H, H-1′, J=5.5 Hz and J=8.7 Hz), 5.5 (m, 1H, H-3′), 4.7 (m, 2H, H-5′,H-5″), 4.5 (m, 1H, H-4′), 2.7 (m, 1H, H-2′), 2.3 (m, 1H, H-2″); FAB<0,(GT) m/e 561 (M−H)⁻, 237 (B)⁻; FAB>0 (GT) 563 (M+H)⁺; [α]_(D) ²⁰+39.05(c=1.05 in DMSO); UV (EtOH 95) υ_(max)=281 nm (ε=9000), υ_(min)=254 nm(ε=4000), υ_(max)=229 nm (ε=31000); Anal. Calcd for C₂₃H₁₉IN₂O₇: C,49.13 H, 3.41 N, 4.98 I, 22.57.

[0186] Found: C, 49.31 H, 3.53 N, 5.05 I, 22.36.

[0187] Reagents:

[0188] Iodine: Fluka, 99.8%, ref 57650

[0189] Cerium ammonium nitrate (CAN): Aldrich, >98.5%, ref 21,547-3

[0190] 3′,5′-Di-O-benzoyl-2′-deoxy-3-N-toluoyl-β-L-thymidine (9)

[0191] To a solution of compound 7 (86.6 g, 0.154 mol) in anhydrouspyridine (1530 ml) containing N-ethyldiisopropylamine (53.6 ml) wasadded, portionwise at 0° C., p-toluoyl chloride (40.6 ml). The reactionmixture was stirred for 2 h at room temperature, then water was added tostop the reaction and the reaction mixture was extracted with methylenechloride. The organic phase was washed with water, dried over sodiumsulfate and evaporated to dryness to give crude3′,5′-di-O-benzoyl-2′-deoxy-3-N toluoyl-5-iodo-β-L-uridine (8) which canbe used for the next step without further purification.

[0192] A solution of the crude mixture 8, palladium acetate (3.44 g),triphenylphosphine (8.0 g) in N-methylpyrolidinone (1375 ml) withtriethylamine (4.3 ml) was stirred at room temperature for 45 min. Then,tetramethyltin (42.4 ml) was added dropwise at 0C under argon. Afterstirring at 100-110° C. overnight, the reaction mixture was poured intowater and extracted with diethyl ether. The organic solution was driedover sodium sulfate and concentrated under reduced pressure. The residuewas purified by a silica gel column chromatography [eluant: stepwisegradient of ethyl acetate (0-10%) in toluene] to give compound 9 as afoam (42.3 g, 48.3% for the 2 steps). ¹H NMR (DMSO) δppm 0.8.3−7.2 (m,15H,2 C₆H₅CO, 1 CH₃C₆H₄CO, H-6), 6.29 (t, 1H, H-1′, J=7.0 Hz), 5.7 (m,1H, H-3′), 4.7−4.5 (m, 3H, H-5′, H^(5″), H-4′), 2.7−2.6 (m, 2H, H-2′,H-2″); FAB<0, (GT) m/e 567 (M−H)⁻, 449 (M−CH₃C₆H₄CO)⁻, 243 (B)⁻, 121(C₆H₅COO)⁻; FAB>0 (GT) 1137 (2M+H)⁺, 569 (M+H)⁺, 325 (M−B)⁻, 245(B+2H)⁺, 119 (CH₃C₆H₅CO)⁻;

[0193] Reagents:

[0194] p-Toluoyl chloride, Aldrich, 98%, ref 10,663-1

[0195] Diisopropylethylamine: Aldrich, >99.5%, ref 38,764-9

[0196] N-methylpyrolidinone: Aldrich, >99%, ref 44,377-8

[0197] Paladium acetate: Aldrich, >99.98%, ref 37,987-5

[0198] Triphenylphosphine: Fluka, >97%, ref 93092

[0199] Tetramethyltin: Aldrich, >99%, ref 14,647-1

[0200] 2′-Deoxy-β-L-thymidine

[0201] A solution of compound 9 (42.3 g, 0.074 mol) in methanolsaturated with ammonia (1850 ml) was stirred at room temperature for twodays. After evaporation of the solvent, the residue was diluted withwater and washed several times with ethyl acetate. The aqueous layer wasseparated, evaporated under reduced pressure and the residue waspurified by a silica gel column chromatography [eluant: stepwisegradient of methanol (0-10%) in methylene chloride] to give pure2′-deoxy-β-L-thymidine (11.62 g, 64.8%) which was crystallized fromethanol; m.p. 185-188° C.; ¹H NMR (DMSO) δppm 11.3 (s, 1H, NH), 7.70 (s,1H, H-6), 6.2 (pt, 1H, H-1′), 5.24 (d, 1H, OH-3′, J=4.2 Hz), 5.08 (t,1H, OH-5′, J=5.1 Hz), 4.2 (m, 1H, H-3′), 3.7 (m, 1H, H-4′), 3.5-3.6 (m,2H, H-5′, H-5″), 2.1−2.0 (m, 2H, H-2′, H-2″); FAB<0, (GT) m/e 483(2M−H)⁻, 349 (M+T−H)⁻; 241 (M−H)⁻, 125 (B)⁻; FAB>0 (GT) 243 (M+H)⁺, 127(B+2H)⁺; )⁺; [α]_(D) ²⁰−13.0 (c=1.0 in DMSO); UV (pH 1) υ_(max)=267 nm(ε=9700), υ_(min)=234 nm (ε=2000).

[0202] Reagent:

[0203] Methanolic ammonia: previously saturated at −5° C., tightlystoppered, and kept in a freezer.

EXAMPLE 6

[0204] Stereoselective Synthesis of 2′-deoxy-β-L-inosine (β-L-dI)

[0205] β-L-dI was synthesized by deamination of 2′-deoxy-β-L-adenosine(β-L-dA) following a procedure previously described in the9-D-glucopyranosyl series (Ref: I. Iwai, T. Nishimura and B. Shimizu,Synthetic Procedures in Nucleic Acid Chemistry, W. W. Aorbach and R. S.Tipson, eds., John Wiley & Sons, Inc. New York, vol. 1, pp. 135-138(1968)).

[0206] Thus, a solution of β-L-dA (200 mg) in a mixture of acetic acid(0.61 ml) and water (19 ml) was heated with sodium nitrite (495 mg), andthe mixture was stirred at room temperature overnight. The solution wasthen evaporated to dryness under diminished pressure. An aqueoussolution of the residue was applied to a column of IR-120 (H⁺)ion-exchange resin, and the column was eluted with water. Appropriatefractions were collected and evaporated to dryness to afford pure β-L-dIwhich was crystallized from methanol (106 mg, 53% yield not optimized):m.p. =209°-211° C.; UV (H₂O), λ_(max)=247 nm; ¹H-NMR (DMSO-d₆)=8.32 and8.07 (2s, 1H each, H-2 and H-8), 6.32 (pt, 1H, H-1; J=6.7 Hz), 4.4 (m,1H, H-3′), 3.9 (m, 1H, H-4′), 3.7−3.4 (m, 2H partially obscured by HOD,H-5′,5″), 2.6 and 2.3 (2m, 1H each, H-2′ and H-2″); mass spectra(mature, glycerol-thioglycerol, 1:1, v/v), FAB>0: 253 (m+H)⁺, 137(base+2H)⁺; FAB<0: 251 (m−H)⁻, 135 (base)⁻; [α]_(D) ²⁰=+19.3 (−c 0.88,H₂O).

[0207] Anti-HBV Activity of the Active Compounds

[0208] The ability of the active compounds to inhibit the growth ofvirus in 2.2.15 cell cultures (HepG2 cells transformed with hepatitisvirion) can be evaluated as described in detail below.

[0209] A summary and description of the assay for antiviral effects inthis culture system and the analysis of HBV DNA has been described(Korba and Milman, 1991, Antiviral Res., 15:217). The antiviralevaluations are performed on two separate passages of cells. All wells,in all plates, are seeded at the same density and at the same time.

[0210] Due to the inherent variations in the levels of bothintracellular and extracellular HBV DNA, only depressions greater than3.5-fold (for HBV virion DNA) or 3.0-fold (for HBV DNA replicationintermediates) from the average levels for these HBV DNA forms inuntreated cells are considered to be statistically significant (P<0.05).The levels of integrated HBV DNA in each cellular DNA preparation (whichremain constant on a per cell basis in these experiments) are used tocalculate the levels of intracellular HBV DNA forms, thereby ensuringthat equal amounts of cellular DNA are compared between separatesamples.

[0211] Typical values for extracellular HBV virion DNA in untreatedcells range from 50 to 150 pg/ml culture medium (average ofapproximately 76 pg/ml). Intracellular HBV DNA replication intermediatesin untreated cells range from 50 to 100 μg/pg cell DNA (averageapproximately 74 pg/μg cell DNA). In general, depressions in the levelsof intracellular HBV DNA due to treatment with antiviral compounds areless pronounced, and occur more slowly, than depressions in the levelsof HBV virion DNA (Korba and Milman, 1991, Antiviral Res., 15:217).

[0212] The manner in which the hybridization analyses are performed forthese experiments result in an equivalence of approximately 1.0 pg ofintracellular HBV DNA to 2-3 genomic copies per cell and 1.0 pg/ml ofextracellular HBV DNA to 3×10⁵ viral particles/ml.

EXAMPLE 7

[0213] The ability of the triphosphate derivatives of β-L-dA, β-L-dC,β-L-dU, β-L-2′-dG, β-L-dI, and β-L-dT to inhibit hepatitis B was tested.Table 1 describes the comparative inhibitory activities of triphosphatesof β-L-dT (β-L-dT-TP), β-L-dC (β-L-dC-TP), β-L-dU (β-L-dU-TP) and β-L-dA(β-L-dA-TP) on woodchuck hepatitis virus (WHV) DNA polymerase, human DNApolymerases α, β, and γ. TABLE 1 WHV DNA pol DNA pol α DNA pol β DNA polγ Inhibitor IC₅₀ K_(i) ^(b) (μM) K_(i) ^(b) (μM) K_(i) ^(b) (μM)β-L-dT-TP 0.34 >100 >100 >100 β-L-dA-TP 2.3 >100 >100 >100 β-L-dC-TP2.0 >100 >100 >100 β-L-dU-TP 8 >100 >100 >100

EXAMPLE 8

[0214] The anti-hepatitis B virus activity of β-L-dA, β-L-dC, β-L-dU,β-L-2′-dG and β-L-dT was tested in transfected Hep G-2 (2.2.15) cells.Table 2 illustrates the effect of β-L-dA, β-L-dC, β-L-dU, and β-L-dTagainst hepatitis B virus replication in transfected Hep G-2 (2.2.15)cells. TABLE 2 Selectivity HBV virions^(a) HBV Ri^(b) Cytotoxicity IndexCompound EC₅₀ (μM) EC₅₀ (μM) IC₅₀ (μM) IC₅₀/EC₅₀ β-L-dT 0.050.05 >200 >4000 β-L-dC 0.05 0.05 >200 >4000 β-L-dA 0.10 0.10 >200 >2000β-L-dI 1.0 1.0 >200 >200 β-L-dU 5.0 5.0 >200 >40

EXAMPLE 9

[0215] The effect of β-L-dA, β-L-dC and β-L-dT in combination on thegrowth of hepatitis B was measured in 2.2.15 cells. The results areprovided in Table 3. TABLE 3 Combination Ratio EC₅₀ L-dC + L-dT 1:3 .023L-dC + L-dT 1:1 .053 L-dC + L-dT 3:1 .039 L-dC + L-dA 11:30 .022 L-dC +L-dA  1:10 .041 L-dC + L-dA 1:3 .075 L-dT + L-dA  1:30 .054 L-dT + L-dA 1:10 .077 L-dT + L-dA 1:3 .035

[0216] Each combination produced anti-HBV activity that was synergistic.In addition, the combination of L-dA+L-dC+L-dT was also synergistic inthis model.

EXAMPLE 10

[0217] The inhibition of hepatitis B replication in 2.2.15 cells byβ-L-DA and β-L-dC, alone and in combination was measured. The resultsare shown in Table 4. TABLE 4 ^(a)β-L-2′-deoxy- ^(b)β-L-2′-deoxy-adenosine (μM) cytidine (μM) % Inhibition ^(c)C.I. 0.5 90 0.05 24 0.0051 0.5 95 0.05 40 0.005 10 0.05 0.05 80 0.34 0.05 0.005 56 0.20 0.050.0005 50 0.56 0.005 0.05 72 0.35 0.005 0.005 54 0.35 0.005 0.0005 300.16 0.0005 0.05 50 0.83 0.0005 0.005 15 0.28 0.0005 0.0005 0 N.A.

EXAMPLE 11

[0218] The efficacy of L-dA, L-dT and L-dC against hepadnavirusinfection in woodchucks (Marmota monax) chronically infected withwoodchuck hepatitis virus (WHV) was determined. This animal model of HBVinfection is widely accepted and has proven to be useful for theevaluation of antiviral agents directed against HBV.

[0219] Protocol:

[0220] Experimental groups (n=3 animals/drug group, n=4 animals/control)Group 1 vehicle control Group 2 lamivudine (3TC) (10 mg/kg/day) Groups3-6 L-dA (0.01, 0.1, 1.0, 10 mg/kg/day) Groups 7-10 L-dT (0.01, 0.1,1.0, 10 mg/kg/day) Groups 11-14 L-dC (0.01, 0.1, 1.0, 10 mg/kg/day)

[0221] Drugs were administered by oral gavage once daily, and bloodsamples taken on days 0, 1, 3, 7, 14, 21, 28, and on post-treatment days+1, +3, +7, +14, +28 and +56. Assessment of the activity and toxicitywas based on the reduction of WHV DNA in serum: dot-blot, quantativePCR.

[0222] The results are illustrated in FIG. 3 and Table 5. TABLE 5Antiviral Activity of LdA, LdT, LdC in Woodchuck Model of Chronic HBVInfection day Control LdA LdT LdC ng WHV-DNA per ml serum^(1,2) 0 381436 423 426 1 398 369 45 123 3 412 140 14 62 7 446 102 6 46 14 392 74 120

[0223] The data show that L-dA, L-dT and L-dC are highly active in thisin vivo model. First, viral load is reduced to undetectable (L-dT) ornearly undetectable (L-dA, L-dC) levels. Second, L-dA, L-dT and L-dC areshown to be more active than 3TC (lamivudine) in this model. Third,viral rebound is not detected for at least two weeks after withdrawal ofL-dT. Fourth, dose response curves suggest that a modes increase in thedose of L-dA and L-dC would show antiviral activity similar to L-dT.Fifth, all animals receiving the drugs gained weight and no drug-relatedtoxicity was detected.

EXAMPLE 12

[0224] Chemical Synthesis of β-L-dC 5′-L-Valyl Ester

[0225] As an illustrative example of the synthesis of β-L-dC aminoesters, β-L-dC 5′-L-valyl ester is synthesized by first protecting theamine group of β-L-dC using (CH₃)₃SiCl. The protected β-L-dC undergoesesterification by the addition of N-Boc L-valine. The ester is thendeprotected to yeild β-L-dC 5′-L-valyl ester. Other methods forsynthesizing amino acid esters are disclosed in U.S. Pat. Nos. 5,700,936and 4,957,924, incorporated herein by reference. The L-valinyl5′-O-ester of L-dA, L-dC, L-dT, and L-dU are preferred embodiments ofthis invention.

[0226] Toxicity of Compounds

[0227] Toxicity analyses were performed to assess whether any observedantiviral effects are due to a general effect on cell viability. Themethod used is the measurement of the effect of β-L-dA, β-L-dC andβ-L-dT on cell growth in human bone marrow clorogenic assays, ascompared to Lamuvidine. The results are provided in Table 6. TABLE 6Compound CFU-GM (μM) BFU-E (μM) β-L-dA >10 >10 β-L-dC >10 >10β-L-dT >10 >10 β-L-dU >10 >10 Lamuvidine >10 >10

[0228] Preparation of Pharmaceutical Compositions

[0229] Humans suffering from any of the disorders described herein,including hepatitis B, can be treated by administering to the patient aneffective amount of a β-2′-deoxy-β-L-erythro-pentofuranonucleoside, forexample, β-L-2′-deoxyadenosine, β-L-2′-deoxycytidine,β-L-2′-deoxyuridine, β-L-2′-deoxyguanosine or β-L-2′-deoxythymidine or apharmaceutically acceptable prodrug or salt thereof in the presence of apharmaceutically acceptable carrier or diluent. The active materials canbe administered by any appropriate route, for example, orally,parenterally, intravenously, intradermally, subcutaneously, ortopically, in liquid or solid form.

[0230] The active compound is included in the pharmaceuticallyacceptable carrier or diluent in an amount sufficient to deliver to apatient a therapeutically effective amount of compound to inhibit viralreplication in vivo, without causing serious toxic effects in thepatient treated. By “inhibitory amount” is meant an amount of activeingredient sufficient to exert an inhibitory effect as measured by, forexample, an assay such as the ones described herein.

[0231] A preferred dose of the compound for all of the abovementionedconditions will be in the range from about 1 to 50 mg/kg, preferably 1to 20 mg/kg, of body weight per day, more generally 0.1 to about 100 mgper kilogram body weight of the recipient per day. The effective dosagerange of the pharmaceutically acceptable prodrug can be calculated basedon the weight of the parent nucleoside to be delivered. If the prodrugexhibits activity in itself, the effective dosage can be estimated asabove using the weight of the prodrug, or by other means known to thoseskilled in the art.

[0232] The compound is conveniently administered in unit any suitabledosage form, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Aoral dosage of 50-1000 mg is usually convenient.

[0233] Ideally the active ingredient should be administered to achievepeak plasma concentrations of the active compound of from about 0.2 to70 μM, preferably about 1.0 to 10 μM. This may be achieved, for example,by the intravenous injection of a 0.1 to 5% solution of the activeingredient, optionally in saline, or administered as a bolus of theactive ingredient.

[0234] The concentration of active compound in the drug composition willdepend on absorption, inactivation, and excretion rates of the drug aswell as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

[0235] A preferred mode of administration of the active compound isoral. Oral compositions will generally include an inert diluent or anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, theactive compound can be incorporated with excipients and used in the formof tablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

[0236] The tablets, pills, capsules, troches and the like can containany of the following ingredients, or compounds of a similar nature: abinder such as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

[0237] The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

[0238] The compound or a pharmaceutically acceptable derivative or saltsthereof can also be mixed with other active materials that do not impairthe desired action, or with materials that supplement the desiredaction, such as antibiotics, antifungals, antiinflammatories, proteaseinhibitors, or other nucleoside or nonnucleoside antiviral agents.Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

[0239] If administered intravenously, preferred carriers arephysiological saline or phosphate buffered saline (PBS).

[0240] In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylacetic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation.

[0241] Liposomal suspensions (including liposomes targeted to infectedcells with monoclonal antibodies to viral antigens) are also preferredas pharmaceutically acceptable carriers. These may be prepared accordingto methods known to those skilled in the art, for example, as describedin U.S. Pat. No. 4,522,81 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

[0242] This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of the thisinvention.

We claim:
 1. A method for treating a host infected with hepatitis Bcomprising administering an effective amount of an anti-HBV biologicallyactive 2′-deoxy-β-L-erythro-pentofuranonucleoside or a pharmaceuticallyacceptable salt or prodrug thereof, wherein the2′-deoxy-β-L-erythro-pentofuranonucleoside has the formula:

wherein R is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl,CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or aphosphate derivative; and BASE is a purine or pyrimidine base which maybe optionally substituted.
 2. The method of claim 1, wherein the2′-deoxy-β-L-erythro-pentofuranonucleoside is β-L-2′-deoxyadenosine or apharmaceutically acceptable salt or prodrug thereof, of the formula:

wherein R is H, mono, di or tri phosphate, amino acid residue acyl, oralkyl, or a stabilized phosphate derivative (to form a stabilizednucleotide prodrug).
 3. The method of claim 1, wherein the2′-deoxy-β-L-erythro-pentofuranonucleoside is β-L-2′-deoxycytidine orpharmaceutically acceptable salt or prodrug thereof of the formula:

wherein R is H, mono, di or tri phosphate, amino acid residue, acyl, oralkyl, or a stabilized phosphate derivative (to form a stabilziednucleotide prodrug).
 4. The method of claim 1, wherein the2′-deoxy-β-L-erythro-pentofuranonucleoside is β-L-2′-deoxyuridine orpharmaceutically acceptable salt or prodrug thereof of the formula:

wherein R is H, mono, di or tri phosphate, amino acid residue, acyl, oralkyl, or a stabilized phosphate derivative (to form a stabilziednucleotide prodrug).
 5. The method of claim 1, wherein the2′-deoxy-β-L-erythro-pentofuranonucleoside is β-L-2′-deoxyguanosine orpharmaceutically acceptable salt or prodrug thereof of the formula:

wherein R is H, mono, di or tri phosphate, amino acid residue, acyl, oralkyl, or a stabilized phosphate derivative (to form a stabilizednucleotide prodrug).
 6. The method of claim 1, wherein the2′-deoxy-β-L-erythro-pentofuranonucleoside is β-L-2′-deoxyinosine orpharmaceutically acceptable salt or prodrug thereof of the formula:

wherein R is H, mono, di or tri phosphate, amino acid residue, acyl, oralkyl, or a stabilized phosphate derivative (to form a stabilizednucleotide prodrug).
 7. The method of claim 1, wherein the2′-deoxy-β-L-erythro-pentofuranonucleoside is β-L-thymidine or apharmaceutically acceptable salt or prodrug thereof of the formula:

wherein R is H, mono, di or tri phosphate, amino acid residue, acyl, oralkyl, or a stabilized phosphate derivative (to form a stabilizednucleotide prodrug).
 8. A method for treating a host infected withhepatitis B comprising administering an effective amount of two or moreanti-HBV biologically active 2′-deoxy-β-L-erythro-pentofuranonucleosidesor a pharmaceutically acceptable salt or prodrug thereof in combinationor alternation, wherein the 2′-deoxy-β-L-erythro-pentofuranonucleosideshave the formula:

wherein R is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl,CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or aphosphate derivative; and BASE is a purine or pyrimidine base which maybe optionally substituted.
 9. A method for treating a host infected withhepatitis B comprising administering an effective amount of an anti-HBVbiologically active 2′-deoxy-β-L-erythro-pentofuranonucleoside or apharmaceutically acceptable salt or prodrug thereof in combination oralternation with an additional anti-hepatitis B agent, wherein the2′-deoxy-β-L-erythro-pentofuranonucleoside has the formula:

wherein R is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl,CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or aphosphate derivative; and BASE is a purine or pyrimidine base which maybe optionally substituted.
 10. The method of claim 9, wherein theadditional anti-hepatitis B agent is selected from the group consistingof 3TC, FTC, L-FMAU, DAPD, famciclovir, penciclovir, BMS-200475, bisporn PMEA (adefovir, dipivoxil); lobucavir, ganciclovir, and ribavarin.11. A compound or pharmaceutically acceptable salt or prodrug thereof ofthe formula:

wherein R is H, mono, di or tri phosphate, amino acid residue, acyl, oralkyl, or a stabilized phosphate derivative (to form a stabilizednucleotide prodrug).
 12. A compound or pharmaceutically acceptable saltor prodrug of claim 11 wherein R is L-valinyl.
 13. A pharmaceuticalcomposition comprising an effective amount of a compound of claim 11 incombination with a pharmaceutically acceptable carrier.
 14. The methodof claim 1, wherein the amino acid is L-valinyl.
 15. The method of claim3, wherein the amino acid is L-valinyl.
 16. The method of claim 4,wherein the amino acid is L-valinyl.
 17. The method of claim 5, whereinthe amino acid is L-valinyl.
 18. The method of claim 6, wherein theamino acid is L-valinyl.
 19. The method of claim 7, wherein the aminoacid is L-valinyl.
 20. The method of claim 8, wherein the amino acid isL-valinyl.
 21. The method of claim 9, wherein the amino acid isL-valinyl.